- Manuals
- Brands
- Agilent
- Software
- CytoGenomics 5.3
- User Guide
Agilent CytoGenomics 5.3 Software User Guide PDF
en
Summary of Content for Agilent CytoGenomics 5.3 Software User Guide PDF
Agilent CytoGenomics 5.2
Agilent Feature Extraction for CytoGenomics
User Guide For Research Use Only. Not for use in diagnostic procedures.
Agilent Technologies
Notices Agilent Technologies, Inc. 2021
No part of this manual may be reproduced in
any form or by any means (including elec-
tronic storage and retrieval or translation
into a foreign language) without prior agree-
ment and written consent from Agilent
Technologies, Inc. as governed by United
States and international copyright laws.
Manual Part Number G1662-90068
Edition Revision A0, May 2021
Agilent Technologies, Inc.
5301 Stevens Creek Blvd.
Santa Clara, CA 95051
Warranty The material contained in this docu- ment is provided as is, and is sub- ject to being changed, without notice, in future editions. Further, to the max- imum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a par- ticular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connec- tion with the furnishing, use, or per- formance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the sep- arate agreement shall control.
Technology Licenses The hardware and/or software described in
this document are furnished under a license
and may be used or copied only in accor-
dance with the terms of such license.
Restricted Rights Legend U.S. Government Restricted Rights. Soft-
ware and technical data rights granted to
the federal government include only those
rights customarily provided to end user cus-
tomers. Agilent provides this customary
commercial license in Software and techni-
cal data pursuant to FAR 12.211 (Technical
Data) and 12.212 (Computer Software) and,
for the Department of Defense, DFARS
252.227-7015 (Technical Data - Commercial
Items) and DFARS 227.7202-3 (Rights in
Commercial Computer Software or Com-
puter Software Documentation).
Safety Notices
CAUTION
A CAUTION notice denotes a haz-
ard. It calls attention to an operat-
ing procedure, practice, or the like
that, if not correctly performed or
adhered to, could result in damage
to the product or loss of important
data. Do not proceed beyond a
CAUTION notice until the indicated
conditions are fully understood and
met.
WARNING
A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly per- formed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated condi- tions are fully understood and met.
Patents Portions of this product may be covered
under US patent 6571005 licensed from the
Regents of the University of California.
Technical Support For US and Canada
Call 800-227-9770 (option 3, 5, 3)
Or send an email to
informatics_support@agilent.com
For all other regions
Agilents world-wide Sales and Support
Center contact details for your location can
be obtained at
www.agilent.com/en/contact-us/page.
2 Feature Extraction for CytoGenomics 5.2 User Guide
In This Guide
Feature Extraction for CytoGenomics
This User Guide shows you how to set up and run the Feature Extraction for CytoGenomics program automatically for a batch of image files and how to extract image files in real time.
1
This chapter introduces the Feature Extraction analysis process and the tasks you can perform with the software to help you with your microarray experiments.
2
This chapter provides instructions on how to set up and run Feature Extraction on a batch of existing Agilent and/or non- Agilent image files. It also shows you how to set up and run Feature Extraction on Agilent image files as they are saved from the scanner in real time.
3
Automatic extraction can take place because grid templates and grid files can be assigned to an image file. This chapter shows you how to create grid files and templates.
4
Another reason why automatic extraction can take place is because a set of Feature Extraction parameters can be assigned to an image file as a protocol. This chapter gives instructions on how to change the parameter values for each step of the Feature Extraction process.
5
Learn how to work with image displays in this chapter. This chapter also includes instructions on working with the frames and panes of the interface display.
5.2 User Guide 3
Acknowledgments
4
Apache acknowledgment
Part of this software is based on the Xerces XML parser, Copyright (c) 1999- 2000 The Apache Software Foundation. All Rights Reserved (www.apache.org).
JPEG acknowledgment
This software is based in part on the work of the Independent JPEG Group. Copyright (c) 1991- 1998, Thomas G. Lane. All Rights Reserved.
Loess/Netlib acknowledgment
Part of this software is based on a Loess/Lowess algorithm and implementation. The authors of Loess/Lowess are Cleveland, Grosse and Shyu. Copyright (c) 1989, 1992 by AT&T. Permission to use, copy, modify and distribute this software for any purpose without fee is hereby granted, provided that this entire notice is included in all copies of any software that is or includes a copy or modification of this software and in all copies of the supporting documentation for such software.
THIS SOFTWARE IS BEING PROVIDED AS IS, WITHOUT ANY EXPRESS OR IMPLIED WARRANTY. NEITHER THE AUTHORS NOR AT&T MAKE ANY REPRESENTATION OR WARRANTY OF ANY KIND CONCERNING THE MERCHANTABILITY OF THIS SOFTWARE OR ITS FITNESS FOR ANY PARTICULAR PURPOSE.
Stanford University School of Medicine acknowledgment
Non- Agilent microarray image courtesy of Dr. Roger Wagner, Division of Cardiovascular Medicine, Stanford University School of Medicine
This software contains material that is Copyright (c) 1994- 1999 DUNDAS SOFTWARE LTD., All Rights Reserved.
Feature Extraction for CytoGenomics 5.2 User Guide
Feature Extraction for CytoGenomics
LibTiff acknowledgement
Part of this software is based upon LibTIFF version 3.8.0.
Copyright (c) 1988- 1997 Sam Leffler Copyright (c) 1991- 1997 Silicon Graphics, Inc.
Permission to use, copy, modify, distribute, and sell this software and its documentation for any purpose is hereby granted without fee, provided that (i) the above copyright notices and this permission notice appear in all copies of the software and related documentation, and (ii) the names of Sam Leffler and Silicon Graphics may not be used in any advertising or publicity relating to the software without the specific, prior written permission of Sam Leffler and Silicon Graphics.
THE SOFTWARE IS PROVIDED "AS- IS" AND WITHOUT WARRANTY OF ANY KIND, EXPRESS, IMPLIED OR OTHERWISE, INCLUDING WITHOUT LIMITATION, ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE.
IN NO EVENT SHALL SAM LEFFLER OR SILICON GRAPHICS BE LIABLE FORANY SPECIAL, INCIDENTAL, INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND, OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE, DATA OR PROFITS, WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE, AND ON ANY THEORY OF LIABILITY, ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE.
5.2 User Guide 5
6
1 Working with Feature Extraction for CytoGenomics
Feature Extraction Processes 18
Overview of Software Capabilities 21
Capabilities in the Feature Extraction for CytoGenomics software window 21
Feature Extraction for CytoGenomics algorithms for data analysis 23
Extraction results 24
What you can do to set up and run automatic batch extraction 25
Setup 25
Run extraction 26
What you can do with images before extraction 27
View image and change image display 27
Find grid and spots on the image manually 27
What you can do with protocol algorithms 29
What you can do with the protocol steps 29
What the protocol steps do for you 29
What you can do with .shp files of extracted images 32
What you can do with extraction results 33
2 Extracting Microarrays Automatically
Setting up and editing projects 36
To set up/edit a Standard Project to run existing image files 36
To set up/edit an On-Time project to run image files in real time 38
To create a project 39
To open a project 40
To save a project 41
Changing properties for individual projects 42
To display project properties for individual projects 42
To change general project property settings 45
To select the location for saving and/or sending output files 46
To change the FTP settings to send result files automatically to Rosetta Resolver 48
Feature Extraction for CytoGenomics 5.2 User Guide 7
To set up to automatically assign protocols 49
To select to use a grid file 50
To select an external DyeNorm list file 50
To select to overwrite previous results 51
To select the incoming image folder for On-Time projects 51
To select a time to terminate real-time extractions 51
To turn off the option to automatically extract XDR images 52
Changing default project properties in Preferences 53
To open the Preferences dialog box 53
To display the General default project properties 54
To select to save the Project Run Summary Report 54
To select to overwrite previous results 55
To select a default folder for a new project 55
To display the default project properties for Output and Data Transfer 56
To select the default location for saving and/or sending output files 57
To change the default FTP settings for automatic data transfer 60
To change default run post-processing plug-ins 60
To display the default properties for standard projects 61
To change the default protocol and its location 62
To select to use a grid file 63
To change the Start Run Mode setting 63
To select an external DyeNorm list file 64
To select to create XDR extraction sets automatically 64
To display the on-time default properties 65
To select the default image folder 65
To select a default time to terminate real-time feature extraction 66
To select to automatically extract an XDR image pair 66
Working with extraction sets 67
What is an extraction set? 67
How does the software create an extraction set? 68
To add extraction sets (image files) to the standard project 69
To sort extraction sets 70
8 Feature Extraction for CytoGenomics 5.2 User Guide
To remove an extraction set from the project 71
To display/edit the extraction Set Configuration table 72
Working with grid templates/files 74
Setting up and Changing Grid Templates and Files 75
To show or hide the Grid Template Browser 75
To add or change a grid template to an extraction set 75
To add or change a grid file to an extraction set 76
To add a grid template 76
To open grid mode from an extraction set 77
To display or change grid template properties 77
To add or change the default protocol in the grid template 78
To add or change the default one-color protocol in the grid template 79
To change the dye normalization gene list in the grid template 79
To create a new internal dye normalization gene list for a grid template 80
To edit an internal dye normalization list 84
To import external dye normalization list to create a new internal list 85
To export an internal dye normalization gene list 85
Working with protocols 87
To show or hide the FE Protocol Browser 87
To add a protocol to an extraction set 87
To change the protocol used in an extraction set 87
To display or change protocol properties 88
To import or export a protocol to or from the database 89
Working with QC metric sets 90
To show or hide the QC Metric Set Browser 90
To associate a QC metric set with a protocol 91
To import or export a QC Metric Set to or from the database 91
To view metric set properties 92
Running Feature Extraction 93
To start feature extraction 93
To stop feature extraction 93
Feature Extraction for CytoGenomics 5.2 User Guide 9
To create a new project with remaining extraction sets if run fails 93
Displaying results 95
To display the text result file in Microsoft Excel 95
To display the Project Run Summary Report 97
To display the QC Report 99
To print the QC Report 101
To display visual results 101
3 Creating Grid Files
About grid templates and grid files 108
When to use manual gridding 110
Analyzing the initial grid for an Agilent image 111
To open the current extraction in grid mode 111
If automatic gridding for an Agilent image fails 113
Step 1. Create a new project for failed extraction sets 113
Step 1b. Manually create a new project for failed extraction sets 114
Step 2. Access grid mode from a project extraction set 115
Step 3. Position grids and save grid file 116
Step 4. Set up and rerun Feature Extraction 116
Creating and using a grid file for non-Agilent images overview 117
Step 1. Start grid mode from the non-Agilent image file 117
Step 2. Position grids, locate spots and save grid file 118
Step 3. Extract the image file with its new grid file 119
Starting grid mode for a non-Agilent image 120
To open an image file 120
To change the Portrait/Landscape view of the image 123
To open grid mode for a non-Agilent image 123
Viewing and changing grid geometry 130
Optimizing grid placement using the help wizard 132
To zoom in or out of the views 132
10 Feature Extraction for CytoGenomics 5.2 User Guide
To undo or redo grid position movements 133
Step 1. Get nominal spot spacing manually 134
Step 2. Get nominal subgrid spacing manually 136
Step 3. Adjust the position of the main grid 137
Step 4. Adjust the subgrid 138
Step 5. Adjusting rotation and skew of subgrids 140
Step 6. Save grid file 140
Manual grid adjustments 141
To rotate the position of the main grid 141
To move or rotate the main grid with Grid Adjustment pane 142
To rotate the subgrid 143
To skew the subgrid corner 144
To position subgrids with Grid Adjustment pane 144
Saving a grid file 146
Calculating spot size and spot centroids 147
Locating spot centroids manually 148
To find a spot to adjust 149
To position the spot centroid 151
To ignore spots 152
To undo or redo spot position movements 152
To position spot centroids with the Grid Adjustment pane 153
Refitting a grid 155
Changing default settings for Grid Editing 157
Shortcuts to help you work with grids and find spots 158
4 Changing Protocol Settings
Changing protocol settings 162
Protocol templates 162
To create a new protocol from a protocol template 162
Creating a new protocol from an existing protocol 163
Feature Extraction for CytoGenomics 5.2 User Guide 11
To open a protocol 164
To view or change the protocol properties 165
To change the protocol steps to run 166
To change the settings for each protocol step 170
To save a protocol with a new name 170
Changing Protocol Step 1: Place Grid 172
To access the settings to place a grid 172
To select the microarray format 173
To select a method for placing the grid 175
To select to use enhanced gridding 175
To select to enable background peak shifting 176
To select to use central part of pack for slope and skew calculations 176
To select to use the correlation method to obtain origin x of subgrids 177
Changing Protocol Step 2: Optimize Grid Fit 178
To access the settings to optimize the grid fit 178
To select the grid format 179
To change the settings to iteratively adjust corners when grid format is selected 180
Changing Protocol Step 3: Find Spots 182
To access the settings to find spots when spot format is automatically determined 182
To select the statistical method to reject pixel outliers 183
To select whether to use mean or median for data analysis 184
To enable the Enhance Spot Finding option 184
To access the settings to find spots when spot format is selected 185
To change values to find and size the spots. 186
To select a spot statistics method to define features 187
To select to autoestimate the local radius to define local background 189
Final calculations for protocol step to find and measure spots 190
Propagation of Find Spot errors to other algorithms 190
Changing Protocol Step 4: Flag Outliers 191
To access the settings to flag outliers 191
To change settings to flag population outliers 193
12 Feature Extraction for CytoGenomics 5.2 User Guide
To change settings to flag non-uniform outliers 194
Changing Protocol Step 5: Compute Bkgd, Bias and Error 199
To access the settings to compute background, bias and error 200
To select the background subtraction method 201
To choose to remove the surface trend found in the data 204
To choose to adjust the background globally (2-color) 206
To choose to perform multiplicative detrending 207
To choose to use robust negative control statistics 208
To select the method to estimate the error (Error Model) 208
To choose to use or not use surrogates 212
Propagation of errors from the Compute Bkgd, Bias and Error algorithm to other algorithms 213
To change settings to test the significance of a feature 213
Changing Protocol Step 6: Correct Dye Biases 216
To access the settings to correct dye biases 216
To choose how to select dye normalization list 217
To choose a method to select a set of dye normalization probes 218
To select to omit background population outliers 219
To select to allow positive and negative controls 219
To select signal characteristics 220
To select a normalization correction method 220
Changing Protocol Step 7: Compute Ratios 222
To access the settings to compute ratios 222
To enter the value for the log ratio limit 223
Use of surrogates for log ratio and p-value calculations 223
Changing Protocol Step 8: Calculate Metrics 227
To access the settings to calculate metrics 227
To select to use spikein targets 228
To change the minimum population for replicate statistics 228
To select the grid test format 228
To change the p-value for differential expression 229
To change the percentile value 229
Feature Extraction for CytoGenomics 5.2 User Guide 13
Changing Protocol Step 9: Generate Results 230
To access the settings to generate results 230
To select the type of QC Report 230
To select to generate a single file for the text output 231
To change the compression factor for the JPEG output 231
5 Changing Image Displays
Displaying and saving image files 234
To open an image file 234
To crop an image 235
To save a cropped image to a new file 237
To add or change the New Array Identifier 239
Loading Feature Extraction visual results 240
To view visual results for low-resolution images: 241
To view visual results for high-resolution images (2- by 3-micron or 20-bit): 242
To save an image (original or cropped) for import to another program 245
To display file information 246
Changing image display settings 247
To change image orientation (landscape/portrait) 247
To change the channels that display 248
To change the display color 249
To change between log and linear color scale 252
To change the intensity scale (color display range CDR) 252
To change the magnification (zooming) 255
To change the window size to fit the image 257
To change the extraction results display 258
To change and save histograms and line graphs 259
To change default image viewing settings 263
To change the print settings 265
Hot Key actions for image displays 267
Changing Feature Extraction window displays 269
14 Feature Extraction for CytoGenomics 5.2 User Guide
To move windows and panes 270
To select the layout to use when software starts 271
Feature Extraction for CytoGenomics 5.2 User Guide 15
16 Feature Extraction for CytoGenomics 5.2 User Guide
Agilent CytoGenomics 5.2 Agilent Feature Extraction for CytoGenomics User Guide
1 Working with Feature Extraction for CytoGenomics
Feature Extraction Processes 18
Overview of Software Capabilities 21
What you can do to set up and run automatic batch extraction 25
What you can do with images before extraction 27
What you can do with protocol algorithms 29
What you can do with .shp files of extracted images 32
What you can do with extraction results 33
This chapter gives you an introduction to the process of Feature Extraction for CytoGenomics and lets you know what you can do with the software to optimize the process. It is not necessary to run the Feature Extraction software separately in order to do feature extractions as part of Agilent CytoGenomics workflows.
NOTE Note that Feature Extraction for CytoGenomics is not supported on
Macintosh systems and is not installed with the Macintosh version of the
Agilent CytoGenomics software. To run an analysis workflow, you must
first extract the image file using the standalone Agilent Feature Extraction
program (on a Windows PC), then use the extracted FE file to run a manual
workflow in the Macintosh version of CytoGenomics.
17Agilent Technologies
1 Working with Feature Extraction for CytoGenomics Feature Extraction Processes
Feature Extraction Processes
Feature Extraction consists of two
major processes: image analysis to
place the grid and locate spots and
data analysis to define and
measure spot features for gene
expression.
18
Below is a flow chart that shows the process for producing reliable microarray results using Feature Extraction for CytoGenomics software on Agilent or non- Agilent microarrays scanned on the Agilent Scanner. The data analysis flow starts with removing outlier pixels and ends with transfer of Feature Extraction results into third- party data analysis programs to compare the results of different experiments. This flow chart shows the protocol steps for Feature Extraction using a CGH protocol.
When you load an Agilent microarray file generated by an Agilent scanner into a project, or start a CytoGenomics feature extraction workflow, the software automatically assigns a grid template, based on the array AMADID, and a protocol.
The protocol uses the grid template (array design) to place the grid and optimize the grid fit. It automatically locates the spots for Agilent microarrays.
To locate spots on non-Agilent microarray files generated by the Agilent scanner, you must interactively position the grid and save the grid file, which you must load into the project.
Feature Ex
Working with Feature Extraction for CytoGenomics 1 Feature Extraction Processes
Feature Extraction for CytoGenomics
After the software places the grid and finds spots, it uses the remaining Feature Extraction algorithm parameters contained in the Feature Extraction protocol to carry out data analysis.
Flags features and background regions that are population and non- uniformity outliers.
Computes background, bias, and error and performs corrections for these as specified in the protocol settings.
If the image is 2-color, it performs dye normalization and, for gene expression, calculates a p-value that is a confidence measure of gene differential expression.
Calculates the log ratios of the dye-normalized green and red channel signals, and calculates final error and significance value.
5.2 User Guide
20
1 Working with Feature Extraction for CytoGenomics Feature Extraction Processes
Generates the stats table and calculates QC metrics for the QC Report.
Generates the output files for each extracted image (Text, MAGE, QC Report, etc.) that you selected in the Project Properties. For Agilent CytoGenomics workflows, the output files are designated in the FE Protocol used for the feature extraction.
Feature extraction results are analyzed and reported by the Agilent CytoGenomics program.
Feature Ex
Working with Feature Extraction for CytoGenomics 1 Overview of Software Capabilities
Overview of Software Capabilities
Capabilities in the Feature Extraction for CytoGenomics software window
Feature Extraction for CytoGenomics
Automatic batch extraction
You create projects to contain extraction sets. The extraction set includes the Agilent or non- Agilent image file, a grid template or grid file to locate spots and a protocol to specify the values for the parameters to carry out Feature Extraction data analysis.
A list of supported scans and array formats are listed in the following table. (Note that GenetiSure Pre- Screen microarray images generated by non- Agilent scanners for single cell applications is not supported by Agilent CytoGenomics.)
Table 1 Supported scans and array formats
Array Format Scanner Scan type Resolution Support Project run summary message
Agilent 65um Agilent 16 bit 5um Supported and tested
Agilent 65um Agilent 20 bit 5um Supported and tested
Agilent 30um Agilent 16 bit 3um Supported and tested
Agilent 30um Agilent 20 bit 3um Supported and tested
Agilent 65um Agilent 16 bit 3um Supported but not tested Information message
Agilent 65um Agilent 16 bit 2um Supported but not tested Information message
Agilent 65um Agilent 20 bit 3um Supported but not tested Information message
Agilent 65um Agilent 20 bit 2um Supported but not tested Information message
Agilent 30um Agilent 16 bit 2um Supported but not tested Information message
Agilent 30um Agilent 20 bit 2um Supported but not tested Information message
5.2 User Guide 21
1 Working with Feature Extraction for CytoGenomics Capabilities in the Feature Extraction for CytoGenomics software window
Agilent 65um Agilent 16 bit 10um Not Supported Warning then proceed
Agilent 65um Agilent 20 bit 10um Not Supported Warning then proceed
Agilent 30um Agilent 16 bit 10um Not Supported Warning then proceed
Agilent 30um Agilent 16 bit 5um Not Supported Warning then proceed
Agilent 30um Agilent 20 bit 10um Not Supported Warning then proceed
Agilent 30um Agilent 20 bit 5um Not Supported Warning then proceed
non-Agilent N/A Agilent any any Limited Support Information message
Agilent 30um non-Agilent 2-color 2um Select Support Information message
Agilent 65um non-Agilent 2-color 5um Select Support Information message
Table 1 Supported scans and array formats (continued)
Array Format Scanner Scan type Resolution Support Project run summary message
22
Explanation of support types Not Supported means that no support is provided by Agilent Technologies. Limited Support means that Agilent provides support on a best- effort basis to help you scan microarrays and extract images, however there is no guarantee or commitment. Select Support is provided for Agilent arrays scanned on selected non- Agilent scanners (specifically, the Roche NimbleGen MS 200 scanners, Molecular Devices GenePix scanners, select Innopsys InnoScan scanners, and select Tecan scanners.) Note that when scanning Agilent arrays on a non- Agilent scanner, you may need to manually grid the arrays if auto- gridding fails. See If automatic gridding for an Agilent image fails" on page 113.
Although Feature Extraction for CytoGenomics 5.1 is capable of
NimbleGen, Molecular Devices, Innopsys, and Tecan scanners, Agilent
can only guarantee the quality of the data output when processing
images of Agilent arrays scanned on an Agilent scanner.
Feature Extraction for CytoGenomics 5.2 User Guide
Working with Feature Extraction for CytoGenomics 1 Feature Extraction for CytoGenomics algorithms for data analysis
Feature Extraction for CytoGenomics
When you start an extraction, the software automatically extracts all the sets in the project and produces a project summary report and a QC report for each image. You can also set up a project to automatically extract Agilent microarray images in real time as soon as the files are made available by the Agilent scanner.
Image analysis
Before extraction you can examine the spots on your microarrays visually and compute statistics about the image, using the histogram and line plots provided in the software. You can interactively position grids to find the spots on the microarray and calculate the spot centroids and sizes.
Feature Extraction for CytoGenomics can handle high- resolution images (2- or 3- micron and 20- bit) from the Agilent G2565CA Scanner. To avoid using large amounts of memory to handle the high- resolution images, Feature Extraction for CytoGenomics loads a low- resolution preview image for first viewing.
When you create a cropped image to one- tenth of its original size or less, the program loads the original high- resolution image for the cropped image.
Histogram and line plots are always based on the original high- resolution image, not the preview one.
Feature Extraction for CytoGenomics algorithms for data analysis
For all images, the protocols define features and background regions, remove pixels, and flag features and background regions that may affect the reliability of the results. In addition, protocols set up the extraction to subtract background information from features and make a background adjustment on signals of low intensity.
5.2 User Guide 23
24
1 Working with Feature Extraction for CytoGenomics Extraction results
The software then calculates a reliable log ratio, p- value, and log ratio error for each feature to give you a confidence measure in the measured log ratio.
Extraction results
Feature Extraction for CytoGenomics offers several types of outputs for each microarray extraction:
Project Run Summary report
MAGE (for Rosetta Resolver)
JPEG (for Rosetta Resolver)
Text (normal and .zip format)
Shape
Grid (used in Feature Extraction only)
QC Report
You can export the text results containing all the parameters, statistical calculations and feature results into a spreadsheet.
To compare the results of different microarray experiments, you can also import various output types into GeneSpring, Agilent Genomic Workbench software, and third- party analysis programs. The text results can be saved in full, compact, QC, and minimal formats, and MAGE- ML results can be saved in both full and compact formats. For details on what information is included in these files, see the Agilent Feature Extraction for CytoGenomics Reference Guide.
You can load and display the results to see the outcome of the statistical and flagging algorithms for each feature and background.
Feature Extraction for CytoGenomics 5.2 User Guide
Working with Feature Extraction for CytoGenomics 1 What you can do to set up and run automatic batch extraction
What you can do to set up and run automatic batch extraction
Setup
See the Feature Extraction for
CytoGenomics Quick Start Guide
for definitions of project, extraction
set, grid template and protocol.
Feature Extraction for CytoGenomics
You can perform the following tasks when you set up automatic batch extraction from within the Feature Extraction for CytoGenomics program:
Create projects and save them with new names.
Load image files to create extraction sets. For Agilent image files, the software automatically:
Loads the required grid template (with associated DyeNorm List, if available) and associated default protocol for each extraction set, if these are available in the database.
Drag and drop scan files onto the project.
Type or change the protocol or grid template for the project.
Drag and drop grid templates or protocols onto each extraction set.
Assign a grid file to an extraction set.
Choose the type of result file to save.
Set up to transfer the files via FTP after extraction.
Delete extraction sets from the project.Add protocols, and QC metric sets to the database.
Edit protocols and grid template properties.
Change the order that extraction sets are processed.
Choose to automatically stop Feature Extraction for CytoGenomics after a specified time when extraction is in real- time mode (On- Time Project).
Load image files for supported types.
5.2 User Guide 25
1 Working with Feature Extraction for CytoGenomics Run extraction
Run extraction
26
Start and stop extracting.
Monitor the extraction progress for each extraction.
View a Summary Report of the extraction set (or batch of extraction sets).
Feature Extraction for CytoGenomics 5.2 User Guide
Working with Feature Extraction for CytoGenomics 1 What you can do with images before extraction
What you can do with images before extraction
View image and change image display
For details on how to view and
change image display, see
Chapter 5, Changing Image
Displays.
Feature Extraction for CytoGenomics
Change color and/or scale of the image for easier viewing.
Select rectangular and elliptical regions of interest.
Create histograms and line graphs.
Create images for other programs.
Print the images.
Compute statistics about the image
Line plots
Histogram plots
Type and save barcode information for Agilent images where the barcode reader has failed.
Flip and rotate the image from landscape to portrait mode and vice versa, for scans of third- party arrays (cannot do for high- resolution images).
Find grid and spots on the image manually
For details on manual gridding, see
Chapter 3, Creating Grid Files.
For Agilent images, grid templates are applied automatically. You would not normally need to adjust the grid unless an error indicating a gridding failure appears in the Summary Report for an extraction. For non- Agilent images, you must create a grid file before you can extract the image.
Interactively adjust the grids on the microarray scan.
Interactively adjust the spots on the microarray (for third- party microarray images only).
Calculate the spot size and compare the position of the nominal spot centroid (+) laid down by the grid with the found centroid position (X) for the spot (for third- party microarray images only).
5.2 User Guide 27
28
1 Working with Feature Extraction for CytoGenomics Find grid and spots on the image manually
Move the found centroid position (X) to where you want it on the spot (for third- party microarray images only).
Feature Extraction for CytoGenomics uses these manually adjusted centroid positions to define features rather than those calculated by the Place Grid algorithm.
Find features by typing feature number, gene name, or probe name (for third- party microarray images only). For Agilent arrays, if a shape file is loaded, you can find features by feature number.
In Grid Mode, select spots to be ignored from analysis (for third- party microarray images only).
Feature Extraction for CytoGenomics 5.2 User Guide
Working with Feature Extraction for CytoGenomics 1 What you can do with protocol algorithms
What you can do with protocol algorithms
For details on protocols, see
Chapter 4, Changing Protocol
Settings in this User Guide and the
Agilent Feature Extraction for
CytoGenomics Reference Guide.
Feature Extraction for CytoGenomics
Protocols contain the values for the parameters used by Feature Extraction for CytoGenomics algorithms to calculate intermediate and final results. See Feature Extraction Processes" on page 18.
What you can do with the protocol steps
Select the protocol steps that you want to use.
Change the default settings for each algorithm in the protocols.
Reload default settings after you have changed them.
What the protocol steps do for you
These protocol algorithms operate in the following order.
Place Grid
For any microarray loaded with a grid file that you saved during Grid Mode (i.e. interactive gridding), automatically calculates centroid positions. Any spots that were manually adjusted are left unchanged.
For any microarray, provides two methods of auto- gridding. The default method is to allow some distortion. This lets the grid have a feature spacing somewhat different from the spacing found in the grid template. The second choice is place and rotate only. This assumes that the feature spacing from the grid template is accurate and only lets this grid move to the best location.
5.2 User Guide 29
30
1 Working with Feature Extraction for CytoGenomics What the protocol steps do for you
For any microarray, user can set the Enable Background Peak Shift flag in order to get better gridding. Some variations in the results are expected with and without use of this flag as the grid positions obtained differ.
For any microarray, user can select to use regions near to the center of packs for slope and skew calculations. This option is of use in cases where pack edges have dim spots and are failing to grid. When this is selected, the user can select to have the software to perform one extra step of correlation following the projection data analysis to get the origin.
Optimize Grid Fit
Find Spots
Calculates statistics for the pixel populations of the features and backgrounds, removing outlier pixels.
Calculates signal statistics for inlier pixels.
Flag Outliers
Compute Bkgd, Bias and Error
If you select to subtract the background, subtracts background from the raw signal of each feature using a local or global background method that you select.
Corrects for spatial gradient across each microarray and any dome effects.
Adjusts the subtracted signal for inaccuracies in the noise level estimation.
For all data, also calculates the error based on a selected error model.
Determines if the feature signal is greater and significantly different from background using the error model calculation.
Feature Extraction for CytoGenomics 5.2 User Guide
Working with Feature Extraction for CytoGenomics 1 What the protocol steps do for you
Feature Extraction for CytoGenomics
Can calculate signals and background for low- intensity features using surrogates.
Correct Dye Biases
Automatically determines if a dye normalization gene list exists for the grid template.
Selects to use dye normalization list or generates one internally.
Selects the features for dye normalization based on a feature selection method of your choosing.
Performs dye normalization on the normalization feature set with a curve fit of your choosing. (SNP probes are not included in dye normalization calculations.)
Compute Ratios
Calculates a final error of the log ratio and a significance value (p- value) based on an error model that you selected in the Compute background step.
Calculate Metrics
Generates the stats table and calculates QC metrics for the QC Report.
You must leave this option turned on to generate a complete stats table and QC report. Some values will be missing if this option is turned off.
See the Agilent Feature Extraction for CytoGenomics Reference Guide for details on QC metrics.
Generate Results
Generates for each extracted image the output files (Text, MAGE, QC Report, etc.) that you selected in the Project Properties.
See the Agilent Feature Extraction for CytoGenomics Reference Guide to learn more about this text file.
5.2 User Guide 31
1 Working with Feature Extraction for CytoGenomics What you can do with .shp files of extracted images
What you can do with .shp files of extracted images
32
Show and hide feature information on the image:
Feature number
Gene name
Raw intensities per color channel
Log ratio (rProcessedSignal/gProcessedSignal) for 2- color microarray images
Show or hide outlier features on the image.
Search and display one feature at a time using feature number.
Get reference to annotation results from Help > Reference Guide.
Print the images.
View the pixels used in the calculation of a feature or set of features.
Feature Extraction for CytoGenomics 5.2 User Guide
Working with Feature Extraction for CytoGenomics 1 What you can do with extraction results
What you can do with extraction results
Feature Extraction for CytoGenomics
View a QC report. You can use the QC report to evaluate if the grid was placed correctly (snapshot of four corners of the grid) and to show if the microarray scan data is reliable.
View all the extraction parameters, statistical calculations and feature results created by the Feature Extraction for CytoGenomics software to know how reliable each feature result is.
Export the text file containing the numbers described above to a spreadsheet or a database of your choice.
Automatically export MAGE- ML files to Rosetta Resolver (FTP) or manually load the files into a database of your choice.
5.2 User Guide 33
34
1 Working with Feature Extraction for CytoGenomics What you can do with extraction results
Feature Extraction for CytoGenomics 5.2 User Guide
Agilent CytoGenomics 5.2 Agilent Feature Extraction for CytoGenomics User Guide
2 Extracting Microarrays Automatically
Setting up and editing projects 36
Changing properties for individual projects 42
Changing default project properties in Preferences 53
Working with extraction sets 67
Working with grid templates/files 74
Setting up and Changing Grid Templates and Files 75
Working with protocols 87
Working with QC metric sets 90
Running Feature Extraction 93
Displaying results 95
You set up to extract a microarray image through the use of projects. Projects let you automatically extract one or more image files either as:
A batch of existing Agilent or non- Agilent files that you add to the project (Standard Project).
One Agilent image at a time as scanning completes (On- time Project).
This chapter explains how to set up and edit projects, run feature extraction, and view the results.
For information on how to use the Auto- Processing function of Agilent CytoGenomics to automate Feature Extraction, see the Agilent CytoGenomics help system.
35Agilent Technologies
2 Extracting Microarrays Automatically Setting up and editing projects
Setting up and editing projects
36
You set up to extract a microarray image through the use of projects. Projects let you automatically extract one or more image files either as:
A batch of existing Agilent or non- Agilent files that you add to the project (Standard Project).
One Agilent image at a time as scanning completes (On- time Project).
To set up/edit a Standard Project to run existing image files
You can run Feature Extraction for Agilent or non- Agilent microarrays automatically through the use of standard projects. To each standard project, you add one or more extraction sets, which consist of the image file, a grid template or file and a protocol. The protocol contains all the values and settings for the parameters used in each step of the Feature Extraction process.
To learn more about working with individual images and with the Feature Extraction software window, see Chapter 5, Changing Image Displays.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To set up/edit a Standard Project to run existing image files
Figure 1 Feature Extraction Standard Project Window
If you do not see the QC Metric Set
Browser, click View > QC Metric Set Browser.
Feature Extraction for CytoGenomics
1 Open a Standard Project, if one is not already open.
See Create a Standard project" on page 39.
2 Change Project Properties.
See To display project properties for individual projects" on page 42.
3 Add the image files to make up extraction sets.
See Working with extraction sets" on page 67.
4 View the Extraction Set Configuration tab sheet to make certain all information is correct for each extraction set.
See To display/edit the extraction Set Configuration table" on page 72.
5 For any extraction set with a non- Agilent microarray image, add an appropriate grid file.
5.2 User Guide 37
38
2 Extracting Microarrays Automatically To set up/edit an On-Time project to run image files in real time
See To add or change a grid file to an extraction set" on page 76.
6 For any extraction set with a non- Agilent microarray image, add a protocol.
See To add a protocol to an extraction set" on page 87.
Starting with Feature Extraction 10.5, protocols are linked to QC metric sets. A QC metric set is no longer assigned to the Project Properties tab. Instead, a QC metric set is assigned to a protocol. You can add or change the default QC metric set associated to the protocol.
See Working with QC metric sets" on page 90.
7 Save the project.
See To save a project" on page 41.
To set up/edit an On-Time project to run image files in real time
You can run Feature Extraction for Agilent microarrays in real time as image files are saved after scanning on the Agilent Scanner. The differences between a Standard project and an On- Time project are listed below:
You can set up an On- Time project only for Agilent microarray images.
You do not add image files or extraction sets (image files, grid templates, and protocols) to the On- Time project.
You can type a maximum total processing time and a maximum time between extractions for an On- Time project.
1 Open an On- Time project.
See Create an On- Time project" on page 39.
2 Change Project Properties.
See To select a time to terminate real- time extractions" on page 51.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To create a project
Feature Extraction for CytoGenomics
3 Save the project.
See To save a project" on page 41.
To create a project
Create a Standard project
To create a new standard project:
Click File > New > Standard Project, Or Click the New project file icon.
When you create the next project after FE_Project1, the name is FE_Project2. The name of every project that you create after that follows in numerical order, whether you save FE_ProjectN or not or close FE_ProjectN or not.
Each time you restart the software, the first new project is FE_Project1, if that is the selection in the Preferences dialog box.
Create an On-Time project
Click File > New > On- Time Project.
Notice that the On- Time Project, Figure 2 does not have a Project Explorer and an Extraction Set Configuration tab.
5.2 User Guide 39
2 Extracting Microarrays Automatically To open a project
Figure 2 On-time project properties window
To open a project
40
To open an existing Standard or On- Time project:
1 Click File > Open > Project, Or Click the Open project file icon.
2 Select the project name, and click Open.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To save a project
To save a project
Feature Extraction for CytoGenomics
To save a Standard or On- Time project to a new name:
1 Click File > Save As.
The default name is FE_ProjectN, where N is the number of the currently loaded project.
2 Type the new name, then click Save.
If you are saving the project for the first time, you can click the Save icon and type any name you choose. The default name that appears is FE_ProjectN, where N is the number of the currently loaded project.
The default directory for projects is C:\ProgramData\Agilent\FeatureExtractionCyto\FEProjects
To save a project to its current name:
Click the Save icon.
If the current project has never been saved, the Save As dialog box appears.
5.2 User Guide 41
2 Extracting Microarrays Automatically Changing properties for individual projects
Changing properties for individual projects
If you are changing properties for
every project that you run, you
probably should change the default
properties in the Preferences
dialog box. See Changing default
project properties in
Preferences" on page 53.
42
When you create a new project or open an existing project in the Feature Extraction for CytoGenomics software, you see a Project Properties tab. The default values found on this tab were set in the Preferences dialog box. For each project that you open, you can change these default properties.
To display project properties for individual projects
Project Properties tab for Standard projects
In the main window for Project setup, click the Project Properties tab.
The Project Properties tab for Standard projects with specified extraction sets looks like Figure 3 below.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display project properties for individual projects
Feature Extraction for CytoGenomics
Figure 3 Standard Project Properties tab, expanded [new screen shot]
Project Properties tab for On-Time projects
When you create a new On- Time project or open an existing one, you see the Project Properties tab.
The Project Properties tab for On- Time projects looks like Figure 4 below.
5.2 User Guide 43
2 Extracting Microarrays Automatically To display project properties for individual projects
44
Figure 4 On-Time Project Properties tab, expanded [new screen shot]
The parameters and settings in this tab are the same as those in a standard project with these differences:
No capacity to select an available grid file.
No setting for default protocol.
No capacity to select an external DyeNorm list file.
Capacity to set up a folder for images coming in from the scanner.
See To select the incoming image folder for On- Time projects" on page 51.
Capacity to terminate the run automatically.
See To select a time to terminate real- time extractions" on page 51.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To change general project property settings
Feature Extraction for CytoGenomics
Capacity to automatically extract XDR (extended dynamic range) images.
See To turn off the option to automatically extract XDR images" on page 52.
To change general project property settings
Change to new values
Double- click the cell to the right of the field name.
True changes to False and vice versa. For list selections, each double- click brings up a different choice in the list.
This does not work for check boxes or Browser cells.
Or, click the arrow to the right of the cell, and select from the list.
Reset changed values to default values
At any time, you can change a modified value back to the original default value.
Right- click the cell on the right, and click Reset to Default.
Change the name of the operator
The only general property that you can change is the name of the Operator.
Click the cell next to Operator, and type the name of the operator.
View the Number of Extraction Sets Included
You cannot change this field. If you add more extraction sets than are visible on screen, you can refer to this field to check the number that you have added.
5.2 User Guide 45
2 Extracting Microarrays Automatically To select the location for saving and/or sending output files
To select the location for saving and/or sending output files
See the Feature Extraction for
CytoGenomics Reference Guide for
a listing of all the results that are
generated in the output files.
46
This portion of the Project Properties tab lets you select the ultimate location(s) for the image and result files. See Select the location for output files" on page 46 and Select the local file folder for saved results" on page 48.
Select the location for output files
Click the arrow to the right of the cell next to each of the result or image file types to select one of the following location(s).
Local file only
FTP send only
Both local file and FTP send
Select to save the files both to a local file folder and to an external location.
The following output files are generated and sent to the location(s) you specify:
MAGE result file
This file contains results in the MAGE- ML (XML) data transfer format, which lets you transfer the data into Rosetta Resolver and Array Express databases.
You can select to send a Full Output Package of MAGE- ML data or a Compact (reduced) set of data. And you can choose to send the data in a Compressed (ZIP) format, or not.
If MAGE output is selected, you also have the capacity to set and pass data ownership and permissions to Rosetta Resolver.
Expand the MAGE folder, and expand the Rosetta Resolver Data Access Setting folder to view the following fields:
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To select the location for saving and/or sending output files
If you do not type names for these
settings, no access control
information is entered into the
MAGE-ML file.
Feature Extraction for CytoGenomics
Rosetta Resolver Data Access Settings: User Name Type the name of the user who will own the data transferred to the Rosetta Resolver database.
Rosetta Resolver Data Access Settings: Control Groups Type the names of the groups who have permission to use the data transferred to the Rosetta Resolver database. If there is more than one group, separate their names with a colon. For example, if you want the groups Public and Special to see the incoming extraction information, type public:special.
JPEG image file
You can control the spatial compression of this JPEG output in the protocol. See To change the compression factor for the JPEG output" on page 231 of this guide.
Text result file
Compact mode contains all the
columns needed for loading into
GeneSpring, DNA Analytics, and
Agilent Genomic Workbench.
You can split this file into three files, each containing one of the sections. You can also import this file into GeneSpring data analysis software, either as a Full set of data, a Compact (reduced), QC, or Minimal. For a listing of the columns included in these formats, see Chapter 3 of the Reference Guide.
You can select to save the text result file in .zip format. The default for this selection is False.
Visual Results
Grid
QC Report
5.2 User Guide 47
2 Extracting Microarrays Automatically To change the FTP settings to send result files automatically to Rosetta Resolver
48
Send a Tiff file via FTP
Because Tiff files already are located in a local file folder, you can select to send them to an external location via FTP, or not.
Double- click the cell to the right of FTP Send Tiff File, and select True or False.
Select the local file folder for saved results
Select to save results in the same local folder as the image This folder must be the folder that contains the images in the extraction sets.
Under Local File Folder, double- click the cell next to Same As Image to select True, if necessary.
Select to save results in a local results folder This folder can be any folder that you choose.
1 Double- click the cell next to Same As Image to select False.
2 Click the cell next to Results Folder.
3 Click the Browse button.
4 Select the folder to contain the results.
5 Click OK.
To change the FTP settings to send result files automatically to Rosetta Resolver
If you use the same settings repeatedly, the values specific to your installation should be set up in the Preferences dialog box. See To change the default FTP settings for automatic data transfer" on page 60.
In the Project Properties tab, expand the FTP Setting folder, and type the values for these FTP settings:
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To set up to automatically assign protocols
Machine Destination
Feature Extraction for CytoGenomics
This field is specific to your Rosetta Resolver server name. Please contact your system administrator for the name to use.
Port
User name
Password
File Destination Folder
Scan information is sent to this folder. For MAGE results, the default setting is mage (/import/mage on the server).
Confirm with your system administrator that the FTP account uses the import directory of the server as the default directory for the transfer.
To set up to automatically assign protocols
Select the highest priority location for the default protocol
The software uses the protocol specified at this parameter step to complete an extraction set if a protocol is available in both the grid template and project. For more information on extraction sets, grid templates and protocols, see Working with extraction sets" on page 67.
1 Click the arrow to the right of the cell next to Highest Priority Default Protocol.
2 Select Grid Template Default or Project Default.
Grid Template Default
If selected, the protocol specified in the grid template is assigned to the extraction set. If the grid template contains no protocol, then the software assigns the project default protocol. Use this selection if you add extraction sets to your projects that require different protocols.
5.2 User Guide 49
2 Extracting Microarrays Automatically To select to use a grid file
Project Default
50
If selected, the protocol specified as the project default protocol is assigned to all extraction sets in the project. If none is specified, then the software assigns the grid template default protocol. Use this selection if you typically add extraction sets that use the same protocol, and this protocol is not the default Agilent protocol assigned to the Grid Template.
Change the project default protocol
1 Click the arrow to the right of the cell next to Project Default Protocol.
2 Select a protocol from the list.
If the protocol you want is not shown on the list, you must import the protocol into the database. See To import or export a protocol to or from the database" on page 89.
To select to use a grid file
Double- click on the cell next to Use Grid file if available and select True or False from the list.
True
False
To select an external DyeNorm list file
To learn how to create an external
DyeNorm list from the
DyeNormList Editor, see To create
a new internal dye normalization
gene list for a grid template" on
page 80.
If the project calls for a DyeNorm list and the image is a non- Agilent array, use an external DyeNorm list. This list must have the column names in the following order: Probe Name, Gene Name, and Systematic Name. This is the same list order for a DyeNorm list exported by the DyeNormList Editor.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To select to overwrite previous results
External DyeNorm lists are only
used with non-Agilent microarrays.
For Agilent microarrays, specify the
dye normalization list in the grid
template. See To change the dye
normalization gene list in the grid
template" on page 79.
Feature Extraction for CytoGenomics
1 Click the cell next to External DyeNorm List File.
2 Click the Browse button.
3 Select the .txt file, and click Open.
To select to overwrite previous results
Double- click on the cell next to Overwrite Previous Results and select True or False.
True
False
To select the incoming image folder for On-Time projects
This selection is for On- Time projects only.
1 To assign a folder for incoming images, click the Browse button in the cell to the right of Incoming Image Folder.
2 Select a folder for the incoming images, and click OK.
To select a time to terminate real-time extractions
This task is for On- Time projects only.
1 Expand Project Run Terminating.
2 Set one or both of the following settings to True or False.
5.2 User Guide 51
2 Extracting Microarrays Automatically To turn off the option to automatically extract XDR images
Use Waiting Idle Time Out
52
If True, you can set an Idle Time in hours and/or minutes. The Idle Time is the time between the arrival of one scan file and the arrival of the next scan file into the real- time image folder.
Use Run Duration Time Out
If True, you can set a Duration Time in days, hours and/or minutes. The Duration Time is the time you are willing to wait for the project feature extraction to complete.
3 If you set either or both settings to True, expand the Idle Time or Duration Time folder, if not expanded.
4 Type the times.
To turn off the option to automatically extract XDR images
This task is for On- Time projects only.
Double- click the cell to the right of Automatic XDR Extraction to change the default setting from True to False.
If the setting is True, the paired XDR images from the scanner are extracted in a combined fashion and a single output file set covering a broader range of data is generated.
If the setting False, the two images are extracted separately and two output file sets are generated.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 Changing default project properties in Preferences
Changing default project properties in Preferences
Feature Extraction for CytoGenomics
You set the default project properties in the Preferences dialog box. The defaults that you type or change here appear in the Project Properties tab when you create a new project.
To open the Preferences dialog box
If you want to change the default settings and values that appear in the Project Properties sheet for all projects, you open the Preferences dialog box.
Click Tools > Preferences.
The Preferences dialog box opens to the folder that was last opened.
Figure 5 Preference Settings window
5.2 User Guide 53
2 Extracting Microarrays Automatically To display the General default project properties
To display the General default project properties
54
1 Open the Preferences dialog box. See To open the Preferences dialog box" on page 53.
2 Open the FE Project folder.
3 Select General.
Figure 6 Preference SettingsFE Project General
To select to save the Project Run Summary Report
The Project Run Summary Report contains a history of the extractions run for a specific project. The Project Run Summary Report is initially saved to a temporary .rtf file (YourComputerName_LastBatchReport.rtf) in the directory containing the project files.
This file is overwritten every time an extraction is run on a project and contains only the latest summary report. If you want to save the summary report for each project for later viewing, then you must specify that intention here.
1 Display the General default project properties. See To display the General default project properties" on page 54.
2 Click the cell next to Auto save extracting report.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To select to overwrite previous results
Feature Extraction for CytoGenomics
3 Select from the list.
Always
Never
Prompt
To select to overwrite previous results
1 Display the General default project properties. See To display the General default project properties" on page 54.
2 Double- click on the cell next to Overwrite Previous Results to select True or False.
True
False
To select a default folder for a new project
This option defines the initial folder into which you save a new project in the Save As dialog box.
1 Display the General default project properties. See To display the General default project properties" on page 54.
2 Click on the cell next to Default Folder for New Project.
5.2 User Guide 55
56
2 Extracting Microarrays Automatically To display the default project properties for Output and Data Transfer
3 Select one of these options from the list.
Last accessed project folder
Folder you accessed to save the previous project.
FE Default Project Folder
Folder set by the application. If you let the installer choose the default installation folder, the path of this folder is C:\ProgramData\Agilent\FeatureExtractionCyto\FEProjects
To display the default project properties for Output and Data Transfer
1 Open the Preferences dialog box. See To open the Preferences dialog box" on page 53.
2 Open the FE Project folder.
3 Select Output & Data Transfer.
Figure 7 Preference SettingsOutput & Data Transfer
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To select the default location for saving and/or sending output files
To select the default location for saving and/or sending output files
Feature Extraction for CytoGenomics
Display the default project properties. See To display the default project properties for Output and Data Transfer" on page 56
See the Feature Extraction for
CytoGenomics Reference Guide for
a listing of all the results that are
generated in the output files.
This portion of the Preferences dialog box lets you select the default location(s) for the image and result files. See Select the default location for output files" on page 57 and Select the default local file folder for saved results" on page 59.
Select the default location for output files
Click the arrow to the right of the cell next to each of the result or image file types to select one of the following location(s).
Local file only
FTP send only
Both local file and FTP send
Select to save the files both to a local file folder and to an external location.
The following output files are generated and sent to the location(s) you specify:
MAGE result file
This file contains results in the MAGE- ML (XML) data transfer format, which lets you transfer the data into Rosetta Resolver and Array Express databases.
You can select to send a Full Output Package of MAGE- ML data or a Compact, or reduced, set of data. And you can choose to send the data in a Compressed (ZIP) format, or not.
If MAGE output is selected, you also have the capacity to set and pass data ownership and permissions to Rosetta Resolver.
5.2 User Guide 57
58
2 Extracting Microarrays Automatically To select the default location for saving and/or sending output files
Expand the MAGE folder, and expand the Rosetta Resolver Data Access Setting folder to view the following fields:
If you do not type names for these
settings, no access control
information is entered into the
MAGE-ML file.
Rosetta Resolver Data Access Settings: User Name Type the name of the user who will own the data transferred to the Rosetta Resolver database.
Rosetta Resolver Data Access Settings: Control Groups Type the names of the groups who have permission to use the data transferred to the Rosetta Resolver database. If there is more than one group, separate their names with a colon. For example, if you want the groups Public and Special to see the incoming extraction information, type public:special.
JPEG image file
You can control the spatial compression of this JPEG output in the protocol. See To change the compression factor for the JPEG output" on page 231 of this guide.
Text result file
A Compact data set contains
sufficient columns for use by
Rosetta Resolver, GeneSpring GX,
CGH Analytics or ChIP Analytics
software. See the Feature
Extraction for CytoGenomics
Reference Guide for a list of these
columns.
You can split this file into three files, each containing one of the sections. You can also import this file into GeneSpring data analysis software, either as a Full set of data, Compact (reduced), QC, or Minimal set. You can also choose to create a .zip file containing the text results.
Visual Results
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To select the default location for saving and/or sending output files
Grid
Feature Extraction for CytoGenomics
Choose to save the grid information to grid.csvfiles (for Agilent and non- Agilent grids) and feat.csv files (for non- Agilent grids). It contains the output of all of the grid placement steps (e.g., the spot centroids and spot sizes).
QC Report
Send a Tiff file via FTP
Because Tiff files already are located in a local file folder, you can select to send them to an external location via FTP, or not.
Double- click the cell to the right of FTP Send Tiff File, and select True or False.
Select the default local file folder for saved results
Select to save results in the local image folder This folder must be the folder that contains the images in the extraction sets.
Under Local File Folder, double- click the cell next to Same As Image to select True, if necessary.
Select to save results in a local results folder This folder can be any folder that you choose.
1 Double- click the cell next to Same As Image and select False.
2 Click the cell next to Results Folder.
3 Click the Browse button.
4 Select the folder to contain the results.
5 Click OK.
5.2 User Guide 59
2 Extracting Microarrays Automatically To change the default FTP settings for automatic data transfer
To change the default FTP settings for automatic data transfer
60
1 Display the default project properties for Output and Data Transfer. See To display the default project properties for Output and Data Transfer" on page 56.
2 In the Project Properties tab, expand the FTP Setting folder, and type the values for these FTP settings:
Machine Destination
This field is specific to your Rosetta Resolver server name. Please contact your system administrator for the name to use.
Port
User name
Password
File Destination Folder
Scan information is sent to this folder. For MAGE results, the default setting is mage (/import/mage on the server).
Confirm with your system administrator that the FTP account uses the import directory of the server as the default directory for the transfer.
To change default run post-processing plug-ins
1 Display the default project properties for Output and Data Transfer. See To display the default project properties for Output and Data Transfer" on page 56.
2 In the Project Properties tab, expand the Post Processing Plug Ins folder, and type or select the plug- in you want to run.
The plug- ins are executed independently, so you can run the same or different plug- ins after each event.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display the default properties for standard projects
Run After Each Array
Feature Extraction for CytoGenomics
Select a plug in to run after each array is extracted. All array related arguments are passed to the executable called.
Run After Each Slide
Select a plug in to run after each slide is extracted, where a slide contains 1 or more arrays. Arguments pointing to files relating to the slide are passed to the executable called.
Run After Each Batch
Select a plug in to run after each batch of extractions is completed. Arguments pointing to files relating to the batch are passed to the executable called.
To display the default properties for standard projects
1 Open the Preferences dialog box. See To open the Preferences dialog box" on page 53.
2 Open the FE Project folder.
3 Click Standard Setting.
Figure 8 Preference Settings Standard Project
5.2 User Guide 61
2 Extracting Microarrays Automatically To change the default protocol and its location
To change the default protocol and its location
62
Select the default highest priority location for the default protocol
The software uses the protocol assigned to this location to complete an extraction set if a protocol is available in both the grid template and project. To learn more about extraction sets, grid templates and protocols, see Working with extraction sets" on page 67.
1 Display the default properties for standard projects. See To display the default properties for standard projects" on page 61.
2 Click the arrow to the right of the cell next to Highest Priority Default Protocol.
3 Select Grid Template Default or Project Default.
Grid Template Default
If selected, the protocol specified in the grid template is assigned to the extraction set. If the grid template contains no protocol, then the software assigns the project default protocol. Use this selection if you add extraction sets to your projects that require different protocols.
Project Default
Change the default protocol for all projects
1 Display the default properties for standard projects. See To display the default properties for standard projects" on page 61.
2 Click the cell next to Project Default Protocol.
3 Select a protocol from the list.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To select to use a grid file
To select to use a grid file
Feature Extraction for CytoGenomics
1 Display the default properties for standard projects. See To display the default properties for standard projects" on page 61.
2 Double- click on the cell next to Use Grid file if available to select True or False from the list.
True
False
To change the Start Run Mode setting
1 Display the default properties for standard projects. See To display the default properties for standard projects" on page 61.
Number of extracting threads
This setting is fixed to one thread. You can no longer change the number of extracting threads.
2 Double- click the cell next to Using Last Saved Layout to
The default setting is False. If True is selected, the software saves the layout changes (i.e. placement of panels and browsers) for the Standard Project and reloads this layout the next time you open Feature Extraction for CytoGenomics.
5.2 User Guide 63
2 Extracting Microarrays Automatically To select an external DyeNorm list file
To select an external DyeNorm list file
External DyeNorm lists are only
used with non-Agilent microarrays.
For Agilent microarrays, specify the
dye normalization list in the grid
template. See To change the dye
normalization gene list in the grid
template" on page 79.
64
If the project calls for a dye normalization list and the image is from a non- Agilent array, use an external DyeNorm list. This list must have the column names in the following order: Probe Name, Gene Name, and Systematic Name. This is the same list order for a DyeNorm list exported by the DyeNormList Editor.
To learn how to create an external
DyeNorm list from a DyeNormList
Editor, see To create a new
internal dye normalization gene list
for a grid template" on page 80.
1 Display the default properties for standard projects. See To display the default properties for standard projects" on page 61.
2 Click the cell next to External DyeNorm List File.
3 Select the external dye normalization list .txt file, and click Open.
To select to create XDR extraction sets automatically
The default for this setting is True. When set to True, the software will automatically create one extraction set for the two images (_H and _L) scanned with extended dynamic range (XDR) turned on in the scanner.
1 Display the default properties for standard projects. See To display the default properties for standard projects" on page 61.
2 Double- click the cell to the right of Automatically Create XDR Extraction Settoselect either True or False.
If you want to be able to add just one of the two images to the extraction set and have the second image loaded automatically, follow step 3.
3 Double- click the cell to the right of Search XDR image file pair on name matching onlytoselect either True or False.
True is the default selection. The pair images must be in the same folder for the software to load the second image automatically to the extraction set.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display the on-time default properties
To display the on-time default properties
Feature Extraction for CytoGenomics
1 Open the Preferences dialog box. See To open the Preferences dialog box" on page 53.
2 Open the FE Project folder.
3 Click OnTime Setting.
Figure 9 Preference Settings On-Time Project Properties
To select the default image folder
1 Display the on- time default properties. See To display the on- time default properties" on page 65.
2 Click on the cell next to Incoming Image Folder.
3 Click Browse.
4 Double- click the image folder, and click OK.
5.2 User Guide 65
2 Extracting Microarrays Automatically To select a default time to terminate real-time feature extraction
To select a default time to terminate real-time feature extraction
66
1 Display the on- time default properties. See To display the on- time default properties" on page 65.
2 Expand Project Run Terminating.
3 Set one or both of the following settings to True or False.
Use Waiting Idle Time Out
If True, you can set an Idle Time in hours and/or minutes. The Idle Time is the time between the arrival of one scan file and the arrival of the next scan file into the real- time image folder.
Use Run Duration Time Out
If True, you can set a Duration Time in days, hours and/or minutes. The Duration Time is the time you are willing to wait for the project feature extraction to complete.
4 If you set either or both settings to True, expand the Idle Time or Duration Time folder, if not expanded.
5 Type the times.
To select to automatically extract an XDR image pair
The default setting is True. That is, the software will automatically extract both the High and Low signal images as a single extraction set when the XDR option is turned on in the scanner.
1 Display the on- time default properties. See To display the on- time default properties" on page 65.
2 Double- click the cell to the right of Automatic Extraction of XDR images to change from True to False, or vice versa.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 Working with extraction sets
Working with extraction sets
What is an extraction set?
Feature Extraction for CytoGenomics
To each standard project you add one or more extraction sets, which consist of the image file, a grid template or file and a protocol. The protocol contains the values and settings for the parameters used in each step of the feature extraction process.
Figure 10 Extraction sets in Project Explorer
Extraction set
Image file
Grid template
Protocol
Grid file
extraction set
20-bit single scans replace XDR
dual scans. To determine if a scan
is 20-bit, click the image file info
button, click the Scan Statistics
tab, and view the Saturation Value
(about 777,000 for 20-bit).
Figure 11 XDR extraction image files marked with _H and _L
5.2 User Guide
2 Extracting Microarrays Automatically How does the software create an extraction set?
grid template
68
Grid information from Agilent array design files stored in the database. A grid template includes feature annotation and general geometry about the microarray (number of rows, columns, subgrids, feature spacings), which is used to locate spots before data analysis takes place. Although not specific to any image, grid template information is usually applied to the image for which the template was designed.
grid file
protocol
How does the software create an extraction set?
Step a
AMADID stands for Agilent
MicroArray Design IDentifier.
The first 6 digits of the array design
file indicate the AMADID. Given a
design file 012097_D_20070820,
012097 is AMADID and 20070820
is YYYYMMDD that the design file
was generated.
For most Agilent microarray images, the grid template is automatically assigned, if the grid template is in the database. You may need to assign a grid template/grid file manually if, for example, the Agilent microarray image failed auto- gridding on a previous run.
A grid file must be used for a non- Agilent extraction set or if you had to manually grid the microarray.
Step b
The added protocol is either the default protocol entered with the grid template or specified for the project. You can select which protocol is the first chosen by the software, the protocol in the grid template or the one in
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To add extraction sets (image files) to the standard project
Feature Extraction for CytoGenomics
the project. If the added grid template or file does not contain a protocol, the software uses the default protocol for the project.
To add extraction sets (image files) to the standard project
You can add an image file or extraction set in one of four ways:
Drag and drop the image file onto Project Explorer
Select the extraction set through the FE Project toolbar.
Select the image file through the Edit menu.
Select the image file through the Project Explorer shortcut menu
To add image files through Windows Explorer:
1 Open Windows Explorer.
2 Select the file or files that you intend to add.
3 Drag them to the Project Explorer and then drop them.
You now see a list of extraction sets.
4 Expand the extraction sets.
Figure 12 Project window after adding image files (extraction sets)
To add extraction sets through the toolbar:
1 Click the Add Extraction Set(s) icon, .
2 Select the image files you want to add, then click Open.
5.2 User Guide 69
70
2 Extracting Microarrays Automatically To sort extraction sets
You can select one or more images for extraction. Use the Shift key to select contiguous files or the Ctrl key to select separate files.
To add image files through the Edit menu:
1 Click Edit > Add Extraction Set(s).
2 Select the image files you want to add, then click Open.
To add image files through the Project Explorer shortcut menu:
1 Right- click in Project Explorer.
2 Click Add Extraction.
3 Select the image files you want to add, then click Open.
You can also add extended dynamic range (XDR) extraction sets. These consist of a pair of images, one of high signal and the other of low signal, scanned with the XDR option turned on in the scanner. Feature Extraction for CytoGenomics extracts both images as a set and creates a single combined set of output files with data covering the combined dynamic range of both scans before it moves on to the next extraction set.
You must make sure that the preferences options for creating and extracting an XDR extraction set are turned on. The default selections are True (ON).
To sort extraction sets
If you want to run extraction sets in an order different from their order in Project Explorer, sort them using these instructions or drag and drop the extraction set in any order in Project Explorer.
1 Click Edit > Sort Extraction Sets.
2 Then select from the list By Name, By Image, By Grid or By Protocol.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To remove an extraction set from the project
Feature Extraction for CytoGenomics
The list is sorted alphabetically and numerically for any of the choices.
To remove an extraction set from the project
To delete an extraction set from Project Explorer
1 Select an extraction set in Project Explorer.
You can select only one extraction set in Project Explorer.
2 Right- click Project Explorer, and click Delete.
The extraction set is immediately deleted.
Or
Click the Delete Extraction Set(s) icon, or click Edit > Delete Extraction Set(s).
If an extraction set in Project Explorer is selected, a message appears to ask if you want to delete the single set in Project Explorer. Click Yes.
If an extraction set or sets in the Extraction Set Configuration table is selected, a message appears to ask if you want to delete the extraction sets in the table.
To delete an extraction set or sets from the Extraction Set Configuration table
1 Select an extraction set or sets in the Extraction Set Configuration table.
You can select more than one by holding down the Ctrl key while you click additional extraction sets.
2 Right- click the selected extraction sets, and click Delete Extraction set(s).
The extraction sets(s) are immediately deleted
Or
Click the Delete Extraction Set(s) icon, or click Edit > Delete Extraction set(s).
5.2 User Guide 71
72
2 Extracting Microarrays Automatically To display/edit the extraction Set Configuration table
If an extraction set or sets in the Extraction Set Configuration table are selected, a message appears to ask if you want to delete the extraction sets in the table. Click Yes.
If different extraction sets are selected in both Project Explorer and the Extraction Set Configuration table, a message appears to ask if you want to delete the extraction set in Project Explorer. Click Yes. To delete the selected sets in the Extraction Set Configuration table, you must repeat step 2.
To display/edit the extraction Set Configuration table
1 Click the Extraction Set Configuration tab.
2 Drag the column marker on the header to expand each cell.
Figure 13 Extraction Set Configuration table
The Extraction Set Configuration tab is a table that lets you review your extraction sets all in one place.
Extraction Set Name
The name of the extraction set. This name is automatically set to be the same as that of the scan file.
Grid Name
Click the cell and click the arrow to add or change this template or file from this column.
Double- click the cell to view the Grid Template Properties dialog box.
Protocol Name
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display/edit the extraction Set Configuration table
Feature Extraction for CytoGenomics
Click the cell and click the arrow to add or change the protocol from this column.
Double- click the cell to view the Protocol Editor.
Output Name
Scan File Name
Double- click this name to see the image in the scan file.
Scan File Path
5.2 User Guide 73
2 Extracting Microarrays Automatically Working with grid templates/files
Working with grid templates/files
74
A microarray design is the layout of probes on a single glass slide with a particular format. Each array design contains the biological and control probe groups, as well as a required Agilent quality control grid (grid template). When working in Feature Extraction for CytoGenomics, the Grid Template Browser displays the array designs from which the grid templates are derived. Designs that are displayed in the Grid Template Browser are the default designs that are included with the Agilent CytoGenomics software, or were imported manually from a hard disk location.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 Setting up and Changing Grid Templates and Files
Setting up and Changing Grid Templates and Files
To show or hide the Grid Template Browser
Feature Extraction for CytoGenomics
When you first open the Feature Extraction for CytoGenomics software after installation, the Grid Template Browser appears on the right- hand side of the main window.
If the Browser is closed, you can re- open it:
Click View > Grid Template Browser.
To add or change a grid template to an extraction set
If you add or change an array
design/grid template that does not
match the design file for an Agilent
image, the software highlights the
template in orange in the
Extraction Set Configuration tab to
warn you. Extraction can still be
run on this set, if you did not select
for Feature Extraction for
CytoGenomics to automatically
assign a grid template, but gridding
or annotation may be incorrect.
If you want to add an array design/grid template or use a different grid template with the image file in the extraction set, follow these instructions:
1 Select a grid template from the list of Grid Templates.
2 Drag and drop the grid template onto the extraction set.
The new grid template appears in the extraction set.
5.2 User Guide 75
2 Extracting Microarrays Automatically To add or change a grid file to an extraction set
To add or change a grid file to an extraction set
76
Although you can add a grid file for Agilent microarrays, you must add or create a grid file for non- Agilent microarrays.
1 In the Project Explorer, right- click the second line of the extraction set, and click Select grid file from the menu.
2 Select the .csv file that you want to use with the image file, and click Open.
To add a grid template
Grid templates (design files) cannot be imported from within the Feature Extraction for CytoGenomics program; they must be imported as design files from within the Agilent CytoGenomics program. To add an array design/grid template to the Grid Template Browser from a file:
1 Launch Agilent CytoGenomics 2.7.
2 From the Supporting Files screen, click Import Design.
3 Select the design/grid template filel (format *.xml) that you want to import into the CytoGenomics database.
Once the design is imported into CytoGenomics, it appears in the Grid Template Browser of Feature Extraction for CytoGenomics. You may need to click Tools > Grid Template > Refresh to refresh the Grid Template Browser.
Grid templates are created from either array design files or grid files. They do not exist as objects in the file system.
See the CytoGenomics help system for more information on importing design files.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To open grid mode from an extraction set
To open grid mode from an extraction set
Feature Extraction for CytoGenomics
If automatic grid placement fails during extraction, you may want to create and use a grid file in place of the grid template, or edit the grid file, depending on which is associated with the extraction set. Or, you may just want to fine tune a grid placement. You can more easily access grid mode from Project Explorer in configuration mode than from the image display.
For non-Agilent images, you can
also access grid mode from the
image display. See Step 1. Start
grid mode from the non-Agilent
image file" on page 117.
1 In Project Explorer, right- click the grid template or grid file in the extraction set.
2 Click Open Current Extraction in Grid Mode.
The image file for the extraction set appears in grid mode, ready for gridding.
See Chapter 3, Creating Grid Files in this guide to learn how to set up or edit a grid file.
3 If you want to add a Design ID not in the list, type the ID in the Add Design ID field, and click Add.
4 To check the release dates, click Check Latest Release Date, Or
5 To add the most recent array design/grid template to the database, click Update DB with New Release.
If you want to add the latest release(s) to the Feature Extraction database without overwriting the most recent version, clear the check box for Update Grid Template in FE database by replacing the most recent version.
To display or change grid template properties
You can only change grid template
properties such as default
protocols and associated dye
normalization lists. Array design
properties (such as geometry)
cannot be changed.
1 Double- click the grid template/array design whose properties you want to display or change
2 Display the grid template information.
5.2 User Guide 77
78
2 Extracting Microarrays Automatically To add or change the default protocol in the grid template
You can display all of the grid information. You can also change the default protocol, the default 1- color protocol, and the dye normalization gene list.
Figure 14 Grid Template Properties window
To add or change the default protocol in the grid template
1 Double- click the grid template whose protocol you want to add or change.
2 Click the cell to the right of Default protocol, and select the protocol from the list.
If you dont see the protocol you want from the list, you must import the protocol into the database first. See To import or export a protocol to or from the database" on page 89.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To add or change the default one-color protocol in the grid template
To add or change the default one-color protocol in the grid template
Feature Extraction for CytoGenomics
Agilent design files and grid templates are the same for 2- color and 1- color or 2- color experiments. To make sure that the software can use a 1- color protocol with 1- color data, both 2- color and 1- color default protocols are specified. The software uses the 1- color protocol when the 1- color scan file is loaded.
1 Double- click the grid template whose protocol you want to add or change.
2 Click the cell to the right of Default OneColor protocol, and select a 1- color protocol from the list.
If you dont see the protocol you want from the list, you must import the protocol into the database first. See To import or export a protocol to or from the database" on page 89.
To change the dye normalization gene list in the grid template
If the protocol specifies use of a dye normalization gene list and the extraction set includes a grid template, you create or select the gene list in the Grid Template Properties window.
1 Double- click the grid template whose dye normalization gene list you want to add or change.
2 Click the arrow to the right of the cell next to Dye Normalization Gene List.
Figure 15 Dye Normalization Gene List options
5.2 User Guide 79
80
2 Extracting Microarrays Automatically To create a new internal dye normalization gene list for a grid template
3 Select one of the following options:
Dye Normalization
Gene List
This is blank if no DyeNorm Gene Lists are available or if you select Set Empty.
If you previously created an internal dye normalization gene list, or selected an external gene list, select a name from this list. Once selected, you can double- click the gene list name to open the DyeNormList Editor to display or edit the list.
Create New
Browse file
Save As
Set Empty
To create a new internal dye normalization gene list for a grid template
1 Double- click the grid template for which you want to create a dye normalization gene list.
2 Click the cell to the right of Dye Normalization Gene List.
3 Click the arrow and click Create New.
NOTE You can create a dye normalization gene list using the Gene/Probe View,
or using the Chromosome View, but not both.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To create a new internal dye normalization gene list for a grid template
Feature Extraction for CytoGenomics
Figure 16 DyeNorm List EditorGene/Probe View
Add genes to list by search criteria
To streamline the process of selecting genes for the list, you can search for genes according to Probe Name, Gene Name or Systematic Name. SNP probes are not included.
1 If necessary, click the Gene/Probe View tab.
2 Click the box next to Name and type a name for the dye normalization list.
5.2 User Guide 81
82
2 Extracting Microarrays Automatically To create a new internal dye normalization gene list for a grid template
3 Under Criteria, click Search for, and then select Probe Name, Gene Name or Systematic Name as the type of name for the search.
4 Select how you want to use the name in the search. You can search according to the exact name, part of the name, or using a regular expression.
5 Type the name or part of the name for the search.
6 Select to include or exclude results.
7 Click Search.
Name
Features Selected (in %)
The dye normalization list must contain at least 1% of the total features in the array. This indicates the % of the features currently selected for the list.
Grid Template Name
This shows the grid template with which you are associating this dye normalization list.
Contains Name (case sensitive)
If selected, you can search for part of the name, or you can use a regular expression. The case must match exactly. Regular expressions give you advanced search capabilities. For example,
If you want to search all probes you can type the regular expression as .(dot) and it will search all the probes.
NOTE For information on using regular expressions for search, see the following
article:
http://msdn.microsoft.com/en-us/library/k3zs4axe%28VS.80%29.aspx.
Match Exact Name (case insensitive)
If selected, you must type the exact probe name, gene name or systematic name, (depending on the type of name you selected in step 1.) For this search, the case does not need to match.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To create a new internal dye normalization gene list for a grid template
Include Results
Feature Extraction for CytoGenomics
If selected, all the results found are transferred from the Available Genes on Array list to the Selected Genes from Array list.
Exclude Results
Add chromosomes to list
1 Click the Chromosome View tab.
Figure 17 DyeNorm List EditorChromosome View
5.2 User Guide 83
84
2 Extracting Microarrays Automatically To edit an internal dye normalization list
2 Select the chromosome(s) from the Available list, and click Add>>. See Add/remove genes or chromosomes to list interactively" on page 84.
Add/remove genes or chromosomes to list interactively
1 Select the gene(s) or chromosome(s) of interest from the Available list.
For multiple selection, hold down the Shift key and click the first name and then the last name or hold the Ctrl key and click for non- contiguous names.
2 Click Add >>, or
Click <
If you attempt to transfer a large number of genes or chromosomes from list to list, the process can take time.
Save dye normalization gene list to internal file
You may have to refresh the screen
in order to see the name of the new
list in the Grid Template Properties
window.
1 Type a name for the dye normalization list in the Name field.
2 Click Save.
If you want to save the internal dye
normalization list as an external
file, see To export an internal dye
normalization gene list" on
page 85.
When you click Save, a list is created using the Name specified, and is placed in the internal list available to the grid template.
To edit an internal dye normalization list
1 Double- click the grid template whose dye normalization gene list you want to change.
2 Click the cell to the right of Dye Normalization Gene List.
3 Select an existing gene list.
4 Double- click the list to bring up the DyeNormList Editor.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To import external dye normalization list to create a new internal list
Feature Extraction for CytoGenomics
5 Add and remove any genes or chromosomes that you want.
Once an internal dye normalization
gene list is created, you cannot
change it. You must save changes
using a different name.
6 Click Save As.
To import external dye normalization list to create a new internal list
1 Double- click the grid template whose dye normalization gene list you want to add or change.
2 If you want to create an internal list from an external .txt file, click the cell next to Dye Normalization Gene List, and click the down arrow.
3 Click Browse file, select the external .txt file and click OK.
This file must be in a special format: 3 columns and a carriage return with Probe Name, Gene Name and Systematic Name listed in that order. The file must not have a row of column headers.
4 Click the down arrow again, and select imported gene list as the default list.
If you need to make changes to the gene list, see To edit an internal dye normalization list" on page 84.
To export an internal dye normalization gene list
After you create an internal dye normalization gene list file, you can export it as a .txt file to any folder you choose.
1 Double- click the grid template whose dye normalization gene list you want to export.
2 Click the down arrow to the right of the cell next to Dye Normalization Gene List, and select the internal gene list.
5.2 User Guide 85
86
2 Extracting Microarrays Automatically To export an internal dye normalization gene list
3 Click the down arrow again, and click Save As.
4 Select a folder you want to save the file to, rename the file, if necessary, and click Save.
The file is saved as an external .txt file.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 Working with protocols
Working with protocols
To show or hide the FE Protocol Browser
Feature Extraction for CytoGenomics
When you first open the Feature Extraction software after installation, the FE Protocol Browser appears on the right- hand side of the main window.
If the Browser has been closed, you can re- open it:
Click View > FE Protocol Browser.
To add a protocol to an extraction set
By default, the program will automatically determine the protocol to use for the extraction. If you want to manually add a protocol to an extraction set, follow these instructions:
1 Click to select a protocol in the FE Protocol Browser.
2 Hold down the mouse button and drag and drop the protocol onto the third line in the extraction set.
The new protocol appears in the extraction set.
To change the protocol used in an extraction set
If you want to use a different protocol than that associated with the grid template or project, follow these instructions:
1 Select a protocol from the FE Protocol Browser.
2 Hold down the mouse button and drag and drop the protocol onto the extraction set.
The new protocol appears in the extraction set, and replaces the previous protocol.
5.2 User Guide 87
2 Extracting Microarrays Automatically To display or change protocol properties
To display or change protocol properties
88
1 Double- click a protocol in the FE Protocol Browser to open the Protocol Editor.
Figure 18 FE Protocol EditorProtocol Properties
2 Click the Protocol Properties button which shows the General tab.
Protocols and QC metric sets are
linked. Each protocol has a metric
set assigned to it.
You can view information about the protocol. Under the General section, you can change the QC metric set that is associated with the protocol. Under the Steps section, you can remove protocol steps from the extraction run.
3 To change the QC metric set assigned to the protocol, click the cell to the right of Metricset, and select a QC Metric Set or Set empty.
4 To remove a protocol step from the run, clear the check box next to the step.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To import or export a protocol to or from the database
Feature Extraction for CytoGenomics
5 Click Save As to save the changes with a new protocol name.
See Chapter 4, Changing Protocol Settings for more details.
To import or export a protocol to or from the database
To import a protocol to the database:
1 Click Tools > FE Protocol > Import.
Or
Right- click the FE Protocol Browser, and click Import.
2 Select a protocol from the list, and click Open.
To export a protocol from the database:
1 Click Tools > FE Protocol > Export.
OR
Right- click the FE Protocol Browser, and click Export.
2 Select a protocol from the list, and click Save.
A copy of the protocol is exported, but the protocol remains in the database.
NOTE When importing a protocol from a previous version of Feature Extraction
for CytoGenomics (i.e., version 4.0 or earlier), the enhanced gridding
features are disabled by default. For instructions on enabling the
enhancements, see To select to use enhanced gridding" on page 175.
5.2 User Guide 89
2 Extracting Microarrays Automatically Working with QC metric sets
Working with QC metric sets
90
By default, the program calculates a set of default statistics that are displayed in the QC report. QC metric sets give you additional statistics for evaluating the feature extraction process on your microarrays. These statistics also appear in the QC report and in the Stats table of the text results file.
QC metric sets are linked with protocols. Each protocol has a metric set assigned to it. When the protocol is used by the software, the QC metric set is also used to generate metrics about the extraction set.
Agilent- created QC metric sets are installed with the Feature Extraction for CytoGenomics software. You can also create your own QC metrics sets using the Quality tools in Agilent CytoGenomics. When you create a QC metric set in Agilent CytoGenomics, it will automatically be available in the QC Metric Set Browser of Feature Extraction. See To import or export a QC Metric Set to or from the database" on page 91.
If you wish to use your custom QC metric set with a protocol, you must associate it to the protocol. See To associate a QC metric set with a protocol" on page 91.
To show or hide the QC Metric Set Browser
This Browser contains the most recent Agilent QC metric sets after the software is installed. In addition, it shows custom QC metric sets that were created in the Quality tab of Agilent CytoGenomics. To show or hide the QC Metric Set Browser:
Click View > QC Metric Set Browser.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To associate a QC metric set with a protocol
To associate a QC metric set with a protocol
Feature Extraction for CytoGenomics
You associate a QC metric set with a protocol in the FE Protocol Editor.
1 Double- click on the protocol to associate a QC metric set with. This opens the FE Protocol Editor.
2 Click the cell next to Metricset, and click the down arrow.
3 Select a QC metric set from the list.
To import or export a QC Metric Set to or from the database
To import a QC Metric Set to the database:
1 Click Tools > QC Metric Set > Import.
Or
Right- click the QC Metric Set Browser, and click Import.
2 Select a QC Metric Set from the list, and click Open.
To export a QC Metric Set from the database:
1 Click Tools > QC Metric Set > Export.
Or
Right- click on a QC metric set in the QC Metric Set Browser, and click Export.
2 Select a QC metric set from the list, and click Save.
A copy of the QC metric set is exported, but the metric set remains in the database.
5.2 User Guide 91
2 Extracting Microarrays Automatically To view metric set properties
To view metric set properties
92
1 In the QC Metric Set Browser, double- click a metric set you wish to view.
For details on the logic used for
metric set evaluation, see the
Agilent Feature Extraction for
CytoGenomics Reference Guide.
The QC Metric Set Properties for the selected metric set is displayed. Names of all the metrics included with this metric set are displayed, along with threshold values, if provided. For details on default metric set properties, see the Agilent Feature Extraction for CytoGenomics Reference Guide.
Figure 19 Metric set properties
2 Click Close to close the dialog box.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 Running Feature Extraction
Running Feature Extraction
To start feature extraction
Feature Extraction for CytoGenomics
1 Click the Project Run mode On/Off icon in the toolbar, or click Project > Start Extracting.
If you did not save the project, a message appears that asks if you want to save the project. You must save the project in order to run feature extraction.
2 Click Yes.
3 Click Save if you want to save the project to the default FE Project N.fep name, or type a new name, and click Save.
During extraction, information on what steps are performed is displayed in the Running Monitor at the bottom of the window. A Project Run Summary report is generated in the Summary Report tab when the extraction is complete.
To stop feature extraction
Click the Stop Extracting icon, or click Project > Stop.
To create a new project with remaining extraction sets if run fails
If a run fails due to a gridding problem, a message appears and provides an easy way to create a new extraction set containing the failed extraction sets. See Step 1. Create a new project for failed extraction sets" on page 113 for details.
5.2 User Guide 93
94
2 Extracting Microarrays Automatically To create a new project with remaining extraction sets if run fails
If a run fails for whatever reason, you can create a project containing the failed or not yet processed extraction sets left over from the previous project run. The following requirements must be met:
Project must be a Standard FE project.
Project must still be in the Run mode.
Project has just stopped extracting and contains failed extraction set(s) or ones not yet run.
1 Click Project > Create Project for Leftover Set(s).
A message appears asking if you want to switch back to Config mode and continue.
2 Click Yes.
Feature Extraction for CytoGenomics creates a new project with all unfinished extraction sets from the current project. Feature Extraction for CytoGenomics also closes the project and starts the new project in its Config mode.
The name for the newly created project is Parent project name_2.
You must save the new project before you can extract or exit the project.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 Displaying results
Displaying results
An Agilent multiplex array is one
that has multiple Hyb/Wash
chambers for separate samples on
a single slide. These were formerly
referred to as multipack arrays.
Feature Extraction for CytoGenomics
A feature extraction run on any image produces the following files:
A Project Run Summary report .rtf file
Text result .txt files (generated per microarray)
For Agilent arrays, a .pdf QC report (generated per microarray) or .html QC report (if you are running with a temporary license.
A .shp visual result file
NOTE For Agilent multiplex arrays, text & QC reports are generated for each
subarray, but just one .shp and Project Run Summary is produced per
extraction of the image.
This section describes how to view these results.
To display the text result file in Microsoft Excel
The result files are named with the following format:
Single pack: extraction set name_protocol name.
Multiplex (formerly known as Multipack): extraction set name_protocol name_pack number, indicated by row number.
1 Go to the folder specified for containing the results.
2 Right- click the text results file (protocol name on end), and click Open with.
3 Click Microsoft Excel.
The text results will open in the spreadsheet. The following figure shows all three tables (FEPARAMS, STATS, and FEATURE) in the file. You can also choose to create a different text file for each table. See To select to generate a single file for the text output" on page 231 for more information.
5.2 User Guide 95
96
2 Extracting Microarrays Automatically To display the text result file in Microsoft Excel
Figure 20 Portion of text results file in Microsoft Excel
For some microarrays, the results will not all fit in an Excel spreadsheet (only the first 65536 rows in Excel 2003 and earlier). Results can be viewed in GeneSpring or DNA Analytics.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display the Project Run Summary Report
To display the Project Run Summary Report
Feature Extraction for CytoGenomics
When you start the extraction run, the Summary Report tab appears showing the project start time. Once extraction is completed, you can see information about the extraction set in run summary. This summary is automatically saved as a Project Run Summary report that you can access later.
1 Go to the folder containing the project whose report you want to display.
The default folder for Feature
Extraction projects is C:\Program
Files\Agilent\C:\Agilent\Agilent
CytoGenomics Edition
FeatureExtraction\FEProjects.
2 Double- click the file named YourComputerName_ LastBatchReport.rtf to display the last saved report for the project.
Or, double- click the file named FE Project N_timestamp.rtf to display a summary report for a specific run of the project.
Figure 21 Project Run Summary
5.2 User Guide 97
98
2 Extracting Microarrays Automatically To display the Project Run Summary Report
You can open the QC Report by double- clicking the blue QC Report link in the Project Run Summary.
Evaluation of grid placement failures
If you see one of these warnings in the Project Summary Report, check the QC report and the .shp file to confirm the success or failure of the grid placement.
Grid failure error. There are many checks that are protocol dependent. These include number of features not found, number non- uniform local background regions, negative control standard deviation, and %CV of replicated features. Depending upon the thresholds set for these metrics in the protocol, the array may or may not show a grid failure error.
The average difference between the nominal grid location and the spot centroid is greater than 10 microns.
If the grid location does not fit within the image, the software will stop the extraction and produce an error in the Project Run Summary.
If automated extraction fails for an Agilent image
Several options are available to troubleshoot the failure. See If automatic gridding for an Agilent image fails" on page 113 for instructions for a successful grid placement and extraction.
If there is a warning that there is no barcode in the MAGE-ML output
Absence of a barcode may be due to several reasons:
Barcode reader failure
Scanner cannot read the Agilent microarray barcode for some reason.
Scan region set up to scan a non- Agilent microarray does not contain the barcode.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display the QC Report
Feature Extraction for CytoGenomics
For Feature Extraction to load MAGE- ML results files into Rosetta Resolver, they must have a barcode or microarray identifier associated with them. You can add this identifier in the Scan Image Properties dialog box. See To add or change the New Array Identifier" on page 239.
To display the QC Report
The report generated depends on the protocol used, and is specified in the Generate Results settings for the protocol. See Changing Protocol Step 9: Generate Results" on page 230.
1 Go to the folder specified for containing the results.
2 Double- click the QC Report .pdf or .html file.
The QC Report can be generated in
.pdf or .html format. If the Feature
Extraction license is a demo
license, only an .html file is
generated.
You can also access the QC Report from the Project Run Summary. Double- click the blue link next to QC Report. See Figure 21 on page 97.
5.2 User Guide 99
100
2 Extracting Microarrays Automatically To display the QC Report
Figure 22 Streamlined CGH QC ReportPage 1
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To print the QC Report
To print the QC Report
Feature Extraction for CytoGenomics
If the QC report is open in Internet Explorer, follow these instructions:
1 Click File > Page Setup.
2 Make sure that it prints in Portrait mode.
3 Select A4 size paper.
4 Click Printer > Properties, and select for color printing.
5 Exit until you see Page Setup, then close Page Setup.
6 Click File > Print Preview.
7 Adjust the margins to display all the images.
8 Click Print.
You can alternatively use another Internet browser that has a Shrink to Fit option for printing, such as Mozilla Firefox.
To display visual results
After extraction is completed, Feature Extraction generates one visual shape result file (.shp) for each tiff image file (.tif). For multiplex Agilent microarrays (more than one microarray on a single glass slide), a single .shp file is for the entire tiff image and covers all the arrays on the slide.
The shape file is only viewable in Feature Extraction. It provides visual results about the extracted microarray image. You can view extraction results such as feature cookies, gene names, raw signals, log ratios, outliers, and pixels used to quantify features (available for high- resolution images).
Load the visual results file (.shp) from Project Explorer
1 Right- click a processed extraction set.
2 Click View Visual Result.
The image is loaded, along with its shape file into the Image workspace. If no shape file is present, the menu item is grayed out.
5.2 User Guide 101
102
2 Extracting Microarrays Automatically To display visual results
Load the visual results file (.shp) from the Image workspace
For more details on how to load the
shape file from the Image
workspace, see Loading Feature
Extraction visual results" on
page 240.
1 Click Open Image, select the .tif file, and click Open.
You can also open the image from Project Explorer (right- click the image name and select View Visual Result) or the Extraction Set Configuration tab sheet (double- click the Scan File Name).
2 Click Feature Extraction > Load Visual Result.
3 Select the .shp file associated with the image, and click Open.
Work with the visual results
By default, the when you load a visual results file, Feature Extraction shows the image in View Outliers Only mode, displaying information about outlier features only. To view results for non- outlier features, see View results on low- resolution images:" on page 103, and View results on high- resolution images (2- by 3- micron or 20- bit):" on page 103.
Figure 23 View Extraction ResultsDefault Settings
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display visual results
Feature Extraction for CytoGenomics
View results on low-resolution images:
1 After .shp file is loaded to the image, zoom in until you see enough of the spots to help you evaluate the results.
2 Click the cropping mode icon to turn off crop mode.
The cursor turns from the cropping cursor to the arrow cursor.
3 Click View > Extraction Results, and clear the View Outlier Only check box.
4 On the image, pass cursor to feature of interest and hold for a few seconds.
A description of the feature appears.
To find a specific feature, use Ctrl-F.
Type the Feature number of the
specific feature you want to find.
Figure 24 Visual results for extracted microarray image
View results on high-resolution images (2- by 3- micron or 20-bit):
After you first load the image, you, in fact, are viewing a preview image. You can only view the original high- fidelity image if you crop the size of the image to one- tenth or less of the original size.
1 After .shp file is loaded to the image, zoom in until you see enough of the spots to help you identify the region of interest.
2 Click the left mouse button at the point where you want to start forming a rectangle for cropping.
5.2 User Guide 103
104
2 Extracting Microarrays Automatically To display visual results
3 With the crop cursor, draw a rectangle equal to or less than one- tenth the size of the image.
Note that the crop cursor is black if the size is within one- tenth (200% to 1000%) of the original size, but it turns red if you are drawing outside the high fidelity range.
4 Then release the mouse button.
Note the status bar at the bottom of the window. It should now read High Fidelity.
5 Click View > Extraction Results, and clear the check box View Outlier Only.
6 On the image, pass cursor to feature of interest and hold for a few seconds.
A description of the feature appears like in Figure 24 on page 103.
View Pixels Used
This option is available for low- resolution and high- resolution images that are in High Fidelity mode (i.e. when the image is zoomed in to one- tenth of its original size).
The View Pixels Used option displays the pixels used to quantify the feature signal within the cookie area. Pixels that are determined to be outliers are shown in black.
1 Complete steps 1- 5 from View results on high- resolution images (2- by 3- micron or 20- bit):" on page 103.
2 Under View > Extraction Results, select View Pixels Used.
Feature Extraction for CytoGenomics 5.2 User Guide
Extracting Microarrays Automatically 2 To display visual results
Feature Extraction for CytoGenomics
Figure 25 View Pixels Used results on a high-resolution image in
high-fidelity mode
5.2 User Guide 105
106
2 Extracting Microarrays Automatically To display visual results
Feature Extraction for CytoGenomics 5.2 User Guide
Agilent CytoGenomics 5.2 Agilent Feature Extraction for CytoGenomics User Guide
3 Creating Grid Files
About grid templates and grid files 108
When to use manual gridding 110
Analyzing the initial grid for an Agilent image 111
If automatic gridding for an Agilent image fails 113
Creating and using a grid file for non-Agilent images overview 117
Starting grid mode for a non-Agilent image 120
Viewing and changing grid geometry 130
Optimizing grid placement using the help wizard 132
Manual grid adjustments 141
Saving a grid file 146
Calculating spot size and spot centroids 147
Locating spot centroids manually 148
Refitting a grid 155
Changing default settings for Grid Editing 157
Shortcuts to help you work with grids and find spots 158
Agilent Agilent Feature Extraction for CytoGenomics software uses grid templates and grid files to determine where spots are located on a microarray before extraction.
This chapter shows you how to troubleshoot failed Agilent image extractions and create grid files for Agilent and non- Agilent images.
Each section of this chapter describes the actions you take to properly position a main grid, subgrids, and ultimately the spot centroids. The section also gives you background information to help you position these grids and spot centroids.
107Agilent Technologies
3 Creating Grid Files About grid templates and grid files
About grid templates and grid files
A grid template contains spacing
information for the specified format
as well as a unique set of probes
and annotations for the microarray
design that it matches.
108
Agilent Feature Extraction for CytoGenomics automates the process of locating microarray spots through the use of grid templates or grid files, which contain spacing information required to place grids for specific Agilent microarray designs. Grid templates are derived from array design files of the same name that reside in the shared Feature Extraction/Agilent CytoGenomics database and are displayed in the Feature Extraction Grid Template Browser. When you import, add, or update a grid template, you actually import, add, or update the entire array design file.
A grid file is similar to a grid
template, except it contains
information specific for a single
scan of a given microarray slide.
If gridding fails for a particular Agilent microarray, you may have to create an Agilent grid file to use for the extraction.Agilent grid files are saved using the image name followed by _grid.csv. If you specify a grid file in a project for extraction of an Agilent image, you must also specify the grid template to be used for the rest of the extraction steps.
For non- Agilent microarrays in a project, you must create an Agilent grid file for the specific non- Agilent microarray you intend to extract. A non- Agilent grid file is stored as a .CSV (comma separated value) file and is not saved in the database. When you save a non- Agilent grid file, two .CSV files are actually created that together define the grid. They are named using the image name followed by _grid.csv or _feat.csv. Both files must be present in order to define the grid.
You cannot, nor do you need to,
adjust spot centroids for Agilent
images because Agilent array
printing is accurate once an initial
grid is found.
When you create a non- Agilent grid file, you must locate the spot centroids, or geometric centers, on the microarrays. To locate spot centroids you interactively position a main grid and subgrids on the microarray. After the subgrids are well- placed, you calculate spot size and centroids.
The software algorithm should locate most, if not all, of the spots where you want them. Any that are incorrect can be moved using a Spot navigator (see Locating spot centroids
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 About grid templates and grid files
Feature Extraction for CytoGenomics
manually" on page 148). This ensures that the Agilent Feature Extraction for CytoGenomics software identifies the correct feature and local background.
5.2 User Guide 109
3 Creating Grid Files When to use manual gridding
When to use manual gridding
110
Table 2 Uses of manual gridding
Image Type Reason to use manual gridding See
Agilent Image Look at how the grid file or template has
placed the grid
Analyzing the initial grid
for an Agilent image" on
page 111
Agilent Image If a gridding error appears in the Summary
Report after Feature Extraction
If automatic gridding for
an Agilent image fails" on
page 113
Non-Agilent Image Create a grid file to be used with the image
for Feature Extraction
Creating and using a
grid file for non-Agilent
images overview" on
page 117
Agilent or non-Agilent image Grid is marked as unplaced in Extraction
Set
Optimizing grid
placement using the help
wizard" on page 132
Non-Agilent Image Evaluation of grid placement Starting grid mode for a
non-Agilent image" on
page 120
non-Agilent image Spot spacing or location of centroids is not
correct
Calculating spot size
and spot centroids" on
page 147
Locating spot centroids
manually" on page 148
Agilent or non-Agilent image Grid placement is not correct after automatic
placement
Manual grid
adjustments" on
page 141
Agilent (rarely) or non-Agilent image Grid does not fit the spots on the grid
(spacing is incorrect in the Geometry)
Refitting a grid" on
page 155
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Analyzing the initial grid for an Agilent image
Analyzing the initial grid for an Agilent image
Feature Extraction for CytoGenomics
When you add an Agilent image to a project, the software assigns a grid template, based on the barcode (AMADID) and a protocol. The protocol uses the grid template to automatically find the spots. Normally, you will not need to do any grid adjustments for an Agilent image, however if you want to display the placement of the grid, or if the grid is labeled as unplaced you can open the extraction in grid mode and make adjustments.
To open the current extraction in grid mode
For Agilent images, the grid
template associated with the
image is automatically assigned to
the extraction set when the grid
template is available in the Grid
Template Browser.
1 Open a project file, or add an extraction to an existing project file.
2 If no grid is open, drag and drop a grid template from the Grid Template Browser, or right click on the grid icon and click Select Grid File to select an existing grid file.
3 Select a protocol.
You must select a protocol that matches the image for grid mode. If not, the grid and spot placement may not be correct.
4 In the Project Explorer, right- click on the extraction set and select Open Current Extraction in Grid Mode.
You can toggle back and forth from
grid mode to the project explorer
from the Window menu on the
toolbar.
The software attempts to place the grid and the image opens in grid mode. In grid mode, you can view the grid placement and make adjustments if necessary.
See Optimizing grid placement using the help wizard" on page 132.
5.2 User Guide 111
112
3 Creating Grid Files To open the current extraction in grid mode
Figure 26 Extraction opened in grid mode
Main Grid View
Main Grid Corner ViewGrid Adjustment pane Help Wizard
Feature Extraction fo
Creating Grid Files 3 If automatic gridding for an Agilent image fails
If automatic gridding for an Agilent image fails
Feature Extraction for CytoGenomics
If the gridding fails during feature extraction, you will see a red error message in the Summary Report that indicates why the gridding failed. See To display the Project Run Summary Report" on page 97 for details on summary reports. If gridding fails, follow the steps below to adjust the grid and rerun an extraction set.
Step 1. Create a new project for failed extraction sets
Step 2. Access grid mode from a project extraction set
Step 3. Position grids and save grid file
Step 4. Set up and rerun Feature Extraction
Step 5. Adjusting rotation and skew of subgrids
Step 1. Create a new project for failed extraction sets
When you attempt to close a project or exit the Feature Extraction software after an extraction where one or more extractions failed due to a gridding error, the software will ask if you want to create a new project containing the failed extraction sets.
Figure 27 Failed extractions due to gridding error
5.2 User Guide 113
114
3 Creating Grid Files Step 1b. Manually create a new project for failed extraction sets
If you click No, the project or Feature Extraction software will close.
If you click Yes, the software will
Create a grid file for all the failed extractions (due to gridding error)
Create a project for all failed extractions (due to gridding error)
Created grids are automatically assigned to their respective extraction sets and their status is changed to unplaced.
The name for the newly created project is Parent project name_2. Every time a new project from leftover sets is created, the software appends _2 to the parent name.
You must save the new project before you can extract or exit the project.
An unplaced grid is one whose x-
and y- coordinates, skew, and
rotation are equal to zero.
When you run the project containing the failed extraction sets, Feature Extraction will check the grid placement status of the first extraction set. If the grid is unplaced a message will appear asking you if you want to open grid mode. Click Yes to open the extraction in grid mode where you can place and save the grid. If you click No, the software will check the next extraction set in the project until all extraction sets have been checked.
Step 1b. Manually create a new project for failed extraction sets
If a run fails for any reason, you can create a project containing the failed or not yet processed extraction sets left over from the previous project run. The following requirements must be met:
Project must be a Standard FE project
Project must still be in the Run mode
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Step 2. Access grid mode from a project extraction set
Feature Extraction for CytoGenomics
Project has just stopped extracting and contains failed extraction set(s) or extraction set(s) not yet run.
1 Select Project > Create Project for Leftover Set(s).
A message appears asking if you want to switch back to Config mode and continue.
2 Click Yes.
Feature Extraction creates a new project with all unfinished extraction sets from the current project. Feature Extraction also closes the current project and starts the new project in Config mode.
The name for the newly created project is Parent project name_2. Every time a new project from leftover sets is created, the software appends _2 to the parent name.
You must save the new project before you can extract or exit the project. To proceed, open the extraction set in grid mode by following Step 2, below.
Step 2. Access grid mode from a project extraction set
This is the quickest way to access grid mode after automatic grid placement has failed. It is the only way to access grid mode for Agilent images.
After gridding, the extraction set
consists of the image file, the grid
template and grid file, and the
protocol.
In the Project Explorer on the left side of the project window, right- click on any object in the extraction set (extraction set name, image, grid template or file, protocol).
3 Select Open Current Extraction in Grid Mode.
The image file for the extraction set appears in grid mode, ready for positioning of the main grid.
Also, the grid file appears under the original grid template in the extraction set.
5.2 User Guide 115
3 Creating Grid Files Step 3. Position grids and save grid file
Step 3. Position grids and save grid file
116
1 Open the extraction in grid mode.
2 Follow the help wizard steps to:
a Position the main grid.
The spot size and spot centroid
options in the Help Wizard are
used for non-Agilent images only.
b Position subgrids, if necessary.
See Optimizing grid placement using the help wizard" on page 132.
The grid is labeled as unplaced in
the Project Explorer until the grid is
positioned and grid mode is closed.
3 Save the grid file.
See Saving a grid file" on page 146.
Step 4. Set up and rerun Feature Extraction
1 Close the grid mode window.
2 Save the project.
3 Click the Project Run mode On/Off icon to start the extraction.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Creating and using a grid file for non-Agilent images overview
Creating and using a grid file for non-Agilent images overview
Feature Extraction for CytoGenomics
Follow these instructions to create and use a grid file for non- Agilent images. You must create a grid file for non- Agilent images to run Feature Extraction.
Step 1. Start grid mode from the non- Agilent image file.
Step 2. Position grids, locate spots and save grid file.
Step 3. Extract the image file with its new grid file.
Step 1. Start grid mode from the non-Agilent image file
1 Open the non- Agilent image file.
See To open an image file" on page 120.
Grid mode cannot be accessed
from an open Agilent image. For
Agilent images, you must access
grid mode from within an
extraction set. See Step 2. Access
grid mode from a project extraction
set" on page 115.
2 Open grid mode and select the gene list type and protocol to use.
See To open grid mode for a non- Agilent image" on page 123.
3 Click OK.
4 In the Geometry Information dialog box, click Auto Fit.
The image file for the extraction set appears in grid mode, ready for positioning of the main window. See Geometry information" on page 129.
5.2 User Guide 117
3 Creating Grid Files Step 2. Position grids, locate spots and save grid file
Step 2. Position grids, locate spots and save grid file
118
1 Follow the help wizard steps in the lower left corner of the grid mode window to:
If you are using a non-Agilent
microarray, the decision to adjust
spots, calculate spot sizes and
centroids, or position spots
manually is based on visual
inspection of how the grid is fitted.
a Determine nominal spot and subgrid spacing manually. (Optional and only for third- party arrays.)
b Determine nominal subgrid spacing. (Optional and for third- party arrays with subgrids only.)
c Position main grid and subgrids, if necessary.
See Optimizing grid placement using the help wizard" on page 132.
The spot size and spot centroid
options are used for non-Agilent
images only.
2 Click the Adjust Spots icon to enable spot location adjustment.
3 Click the Calculate spot size and centroids icon and calculate the spot sizes and centroids.
See Calculating spot size and spot centroids" on page 147.
4 Visually inspect the spot centroids and if necessary, position spots manually.
See Locating spot centroids manually" on page 148.
5 Save the grid file.
See Saving a grid file" on page 146.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Step 3. Extract the image file with its new grid file
Step 3. Extract the image file with its new grid file
Feature Extraction for CytoGenomics
To test the new grid file, create the project and add the extraction set.
1 Select Feature Extraction > Create a New FE Project.
A new Feature Extraction project is created with the image file already added.
2 In the Project Explorer, right- click on the grid icon below the image file, and click Select Grid File.
3 In the Select Grid file for Extraction Set dialog box, select your saved grid file, and then click Open.
4 From the FE Protocol Browser, drag the protocol for the image type to the extraction set.
5 Save the project and run Feature Extraction to see if the grid works.
5.2 User Guide 119
3 Creating Grid Files Starting grid mode for a non-Agilent image
Starting grid mode for a non-Agilent image
120
You can view and adjust grid placement using grid mode. For non- Agilent images, grid mode can be accessed from either an open image file or from an extraction set that has an associated grid and protocol.
To open an image file
You cannot access grid mode for
Agilent images from an open
image. See Step 2. Access grid
mode from a project extraction
set" on page 115.
1 Click on the toolbar Open image file button, or click File > Open > Image, to display the Open Dialog Box.
2 In the Open Dialog Box, double- click on a .tif file from the scanner, or select a file and click Open.
You can also open an image file by
dragging the file to the Feature
Extraction desktop icon.
An image of the scanned microarray contained in the file opens in its own window.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To open an image file
Feature Extraction for CytoGenomics
Figure 28 TIFF image for non-Agilent microarray
Toggle Log Scale Icon
Grid Mode IconZoom In/Zoom Out Icons
5.2 User Guide 121
122
3 Creating Grid Files To open an image file
3 To see the spots more clearly, click the Toggle Log Scale icon and click the Zoom In icon to increase the image size.
Figure 29 Image view with log scale on and image zoomed
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To change the Portrait/Landscape view of the image
To change the Portrait/Landscape view of the image
The software changes orientation
in this way because the Agilent
scanner scans the long side of the
microarray through the glass. This
is an orientation that is flipped and
rotated compared with other
scanners.
Feature Extraction for CytoGenomics
You must change the orientation of any opened image if the gene list file that you intend to use to provide grid information organizes its data with the opposite orientation. You typically do not have to do this for Agilent images, but you usually have to do this for non- Agilent images.
1 Select Tools > Flip Upper Left to Lower Right (Landscape/Portrait).
The software takes the upper left corner of the image and flips it to the lower right no matter if you change the orientation from Portrait to Landscape or vice- versa.
2 Select File > Save modified image to save the image.
To open grid mode for a non-Agilent image
1 Open a non- Agilent image file.
2 Click the Grid button on the image toolbar.
The Create/Edit Grid dialog box opens.
5.2 User Guide 123
124
3 Creating Grid Files To open grid mode for a non-Agilent image
Figure 30 Create/Edit Grid dialog box
3 Select the Gene List Type to let you position the grid.
If you select a file whose data orientation is different from that of the image, the Agilent software will not be able to set the grid properly. Cancel out of this dialog box and change the orientation if you have to.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To open grid mode for a non-Agilent image
Agilent Grid File (*_grid.csv)
Feature Extraction for CytoGenomics
This is a grid file that has been saved from the previous creation of a grid on an image.
If you want to edit a grid or change the location of the spot centroids for an image, select the grid file previously saved for that image.
If you have never set up an initial grid and the image resembles one whose grid you have set up before, select that grid file from the already existing grid files.
Gene List
The layout information and feature annotations must be in tab- delimited text format and must contain at least these columns: Grid index (or SubGridRow/SubGridCol), SpotRow, SpotCol and GeneName or Probename.
The tab text file can be a GAL file, a results file or an Excel file exported as a tab- delimited text file.
Control Type can also be in the tab text file. See the Feature Extraction for CytoGenomics Reference Guide to see the definitions of the control types that can appear in the tab text file.
No Gene List
4 Select a protocol.
For 2 color Gene Expression, the
default GE2-NonAT_1010_Jun10
protocol provided with the
software can be used for
non-Agilent images.
Select a protocol to help with grid placement and spot finding. Grid placement and spot finding algorithms differ between protocols. You must select a protocol that matches the image for grid mode. If not, the grid and spot placement may not be correct.
5 Click OK.
What happens next will depend on what you have selected for the gene list type:
5.2 User Guide 125
126
3 Creating Grid Files To open grid mode for a non-Agilent image
If you select Agilent Grid File as the gene list type
A Browser appears that lets you select the grid file. The grid file must already exist for the image.
Select a grid file, and click Open.
The Main Grid View appears with the grid approximately positioned.
If you select Gene list and then select a gal file as the gene list type
The Geometry Information dialog box appears for the gal file grid.
1 Change the default values if you need to.
2 Click Auto Fit or Manual Fit.
For a description of all the fields and buttons in the Geometry Information dialog box, see Geometry information" on page 129.
The Main Grid View for this image appears.
If you select Gene list and then select a tab text file as the gene list type
The Tab Text Grid File Preview window appears.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To open grid mode for a non-Agilent image
Feature Extraction for CytoGenomics
Figure 31 Tab Text Grid File Preview window with header selected
1 Click each of the column headers that correspond to the following information fields:
Subgrid Col
Subgrid Row
Spot Col
Spot Row
Probe Name
2 Select the appropriate name from the list.
You must change the names of the columns listed above in order for gridding to work with a tab text file. You can also change other column names, such as Gene Name or Control.
5.2 User Guide 127
128
3 Creating Grid Files To open grid mode for a non-Agilent image
Figure 32 Tab Text Grid File Preview window with headers selected
3 Click OK.
The Geometry Information dialog box appears.
4 Click Auto Fit or Manual Fit.
For a description of all the fields and buttons in the Geometry Information dialog box, see Viewing and changing grid geometry" on page 130.
The Main Grid View for the non- Agilent image appears.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To open grid mode for a non-Agilent image
Feature Extraction for CytoGenomics
If you select No Gene List
The Geometry Information dialog box appears.
1 Change the default values if you need to.
2 Click Auto Fit or Manual Fit.
For a description of all the fields and buttons in the Geometry Information dialog box, see Viewing and changing grid geometry" on page 130.
The Main Grid View for the non- Agilent image appears.
Geometry information
This dialog box appears when you click OK from the Create/Edit Grid dialog box.
If Gene List or No Gene List are selected as the Gene List Type in the Create/Edit Grid dialog box, the Feature Extraction software attempts to discover the grid and will populate the Geometry Information fields with the information from that attempt.
For Gene List, if you select a .gal file, the parameters from the .gal file is displayed in the Geometry Information dialog box.
For details on all of the fields and buttons of the Geometry Information dialog box, see Viewing and changing grid geometry" on page 130.
5.2 User Guide 129
3 Creating Grid Files Viewing and changing grid geometry
Viewing and changing grid geometry
130
The following dialog box appears when you click OK from the Create/Edit Grid dialog box when entering grid mode from a non- Agilent image file (see Starting grid mode for a non- Agilent image" on page 120), or when you Refit a grid (see Refitting a grid" on page 155).
Figure 33 Geometry Information dialog box
Main grid/Array
geometry
For the main grid, enter dimensions in the form of number of columns and rows, or enter the distance between subgrids in pixels for both columns and rows.
Subgrid geometry
For subgrids, enter dimensions in the form of number of columns and rows, or enter the distance between spots in pixels for both columns and rows.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Viewing and changing grid geometry
Double Density Offsets
Feature Extraction for CytoGenomics
The double density offset is applied when the array being gridded contains features consisting of two interlaced rectangular grids. The spacing of these rectangular grids is specified in the Double Density row and column offsets.
Nominal spot diameter
Because spots may not be circular, enter the horizontal and vertical dimensions in pixels.
Reset
Auto Fit
Manual Fit
Cancel
Table 3 Geometry information that you can change on the Geometry Information (GI) dialog box*
* You can change the nominal spot and subgrid spacing in grid mode.
File Type Initial GI dialog box displays?
Change main grid dimensions?
Change subgrid dimensions?
Change spot spacing?
Change spot size?
Agilent Grid No
The Geometry Information dialog box does not appear when you select Agilent Grid File as the gene list type. The columns for the Agilent Grid file type apply to the refit Geometry Information dialog box, which does appear when you attempt to refit an Agilent grid (see Refitting a grid" on page 155).
No No Yes No
GAL Format Yes Yes No Yes Yes
Tab Text Yes Yes No Yes Yes
No gene list Yes Yes Yes Yes Yes
5.2 User Guide 131
3 Creating Grid Files Optimizing grid placement using the help wizard
Optimizing grid placement using the help wizard
Although the Help Wizard is
available for both Agilent and
non-Agilent arrays, some steps
apply only to non-Agilent
(third-party) arrays.
132
Grid mode provides a Help Wizard to step you through the process of optimizing the placement of the grids. To view the help steps, in the Help pane on the lower left of the grid mode window, click Next. To go back to the previous step, click Previous. The help wizard provides instructions for the following tasks:
Step 1. Get nominal spot spacing manually (for non- Agilent arrays only)
Step 2. Get nominal subgrid spacing manually (for non- Agilent arrays only)
Step 3. Adjust the position of the main grid
Step 4. Adjust the subgrid
Step 5. Adjusting rotation and skew of subgrids
Step 6. Save grid file
To zoom in or out of the views
Before you optimize the grid placement, it may be necessary to zoom the image in order to see the spots more easily. There are two ways to zoom the image:
See To change the magnification
(zooming)" on page 255 of this
guide for more details on zooming.
From the Feature Extraction menu bar, select View > Zoom and choose a % zoom from 10% to 1000%. A 10% zoom is defined and you can select a zoom level in 10% increments. You can also change the default zoom settings. See Changing default settings for Grid Editing" on page 157 for details.
On the image toolbar, click one of the zoom icons.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To undo or redo grid position movements
Figure 34 Grid mode view
To undo or redo grid position movements
Main Grid View Click to zoom out.
Corner View
Click to zoom to 100%.
Grid Adjustment pane Help Wizard
Click to zoom in.
Image Toolbar
Feature Extraction for CytoGenomics
Click the Undo button, , to undo an action.
Click the Redo button, , to redo an action.
5.2 User Guide
3 Creating Grid Files Step 1. Get nominal spot spacing manually
Step 1. Get nominal spot spacing manually
134
This step is optional, and is only used for third- party arrays. Perform this step if spacing information for spots are unknown.
1 Make sure that the color log scale has been turned on to view the corner spots on the main grid more easily.
2 On the gridding toolbar, click the View and Measure Image icon .
3 If necessary, zoom the image. See To zoom in or out of the views" on page 132.
4 On this image view (which is appended with [CROPPED] in the image name), click on the center of the top left spot and drag the mouse horizontally to the center of the spot in the next column. (Do not release the mouse button.) Read and note the column distance from the status bar. This is the left- hand number shown in parentheses; for example, (16x0). (You will need this value later.)
5 On the image view, click on the center of the top left spot and drag the mouse vertically to the spot center in the first column of the second row. (Do not release the mouse button.) Read and note the row distance from the status bar. This is the right- hand number shown in parentheses; for example, (0x17). (You will need this value later.)
6 Close the cropped image view.
7 In the grid mode view, click the Grid Definition tab.
8 In the Grid Definition panel, double- click on the space next to Col under Nominal Spot spacing, and enter the column distance value from step 4.
9 In the Grid Definition panel, double- click on the space next to Row under Nominal Spot spacing, and enter the row distance value from step 5.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Step 1. Get nominal spot spacing manually
Feature Extraction for CytoGenomics
Figure 35 Reading column or row distance from image view
Read column (horizontal) distance
Read row (vertical) distance
5.2 User Guide 135
3 Creating Grid Files Step 2. Get nominal subgrid spacing manually
Step 2. Get nominal subgrid spacing manually
136
This step is optional and only for third- party arrays. Perform this step if spacing for the subgrids is unknown.
1 Make sure that the color log scale has been turned on to view the corner spots on the main grid more easily.
2 On the gridding toolbar, click the View and Measure Image icon .
3 If necessary, zoom the image. See To zoom in or out of the views" on page 132.
4 On the newly- opened image, click on the center of the top right spot for the first subgrid and drag the mouse horizontally to the center of the first spot in the adjacent subgrid. (Do not release the mouse button.) Read and note the column distance from the status bar. This is the left- hand number shown in parentheses; for example, (43x0). (You will need this value later.)
5 On the opened image view, click on the center of the bottom left spot of the first subgrid and drag the mouse vertically to the center of the top left spot of the subgrid below. (Do not release the mouse button.) Read and note the row distance from the status bar. This is the right- hand number shown in parentheses; for example, (0x42). (You will need this value later.)
6 Close the opened image view.
7 In the grid mode view, click the Grid Definition tab.
8 In the Grid Definition panel, double- click on the space next to Col under Nominal Subgrid spacing, and enter the column value from step 4.
9 In the Grid Definition panel, double- click on the space next to Row under Nominal Subgrid spacing, and enter the row value from step 5.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Step 3. Adjust the position of the main grid
Step 3. Adjust the position of the main grid
Feature Extraction for CytoGenomics
1 Make sure that the color log scale has been turned on to view the corner spots on the main grid more easily.
2 On the toolbar, click the Adjust Main Grid icon .
3 Move the mouse into the upper Main Grid View until the move cursor appears.
4 Click and drag the move cursor so that the top left corner of the grid is positioned on the top left spot of the microarray image.
5 To do fine adjustment of the main grid, click on the top left spot in the top left pane of the lower Corner View pane.
Note that the thick line represents
the main grid and the thin line
represents the subgrids.
Figure 36 Adjust main grid view
5.2 User Guide 137
3 Creating Grid Files Step 4. Adjust the subgrid
Step 4. Adjust the subgrid
For a microarray with only one grid,
if the main grid is well-positioned,
there is no reason to move the
subgrid. This is the case for most
Agilent microarrays.
138
When you position subgrids you are, in effect, positioning nominal spot centroids (+). These nominal spot centroids are still subject to adjustment by the spot finding algorithm in Feature Extraction. You can position the subgrids such that the nominal spot centroids are just inside the spots. These positions are good enough for the spot finding algorithm to calculate more accurate spot centroids.
1 On the toolbar, click the Adjust Subgrid icon.
2 In the Main Grid View, click on a subgrid.
3 In the Main Grid View, click and drag the top left corner of the subgrid to the top left spot of the subgrid.
4 For fine adjustment of the grid, click the Select Corners and Automatically Fit Grid icon on the toolbar to turn it off.
5 In lower Corner View, click the center of the top left spot of the selected subgrid.
6 Repeat step 2 through step 5 for the remaining subgrids.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Step 4. Adjust the subgrid
Feature Extraction for CytoGenomics
Figure 37 Adjust subgrid view
Subgrid Main Image View Subgrid Corner View
5.2 User Guide 139
3 Creating Grid Files Step 5. Adjusting rotation and skew of subgrids
Step 5. Adjusting rotation and skew of subgrids
140
1 In the Main Grid View, click on a subgrid to select it.
When Select Corners and Automatically Fit Grid is enabled,
the software will automatically
calculate a fit based on three
user-selected corner points.
2 On the toolbar, click to select the Select Corners and Automatically Fit Grid icon, if it is not on.
3 In the lower view, if the corners of the grid are not near to the corner spots of the image, adjust the grid location by clicking and dragging the grid in the bottom right pane of the lower four corner view.
4 Move the cursor to the top left pane of the lower Corner View. Notice the cursor turns to a red cross. Click on the top left spot of the subgrid.
5 In the top right pane of the lower Corner View, click on the top right spot (in the first row) of the subgrid.
6 In the bottom left pane of the lower Corner View, click on the lower left spot (in the first column) of the subgrid.
7 Repeat step 3 through step 6 for the remaining subgrids.
Step 6. Save grid file
1 Click the Save Grid button on the main toolbar.
2 The grid is saved to a ImageName_grid.csv file where ImageName is the name of the image associated with the grid, and the directory containing the grid file is the same as the directory of the original image. A features file is saved at the same time as a ImageName_feat.csv file.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Manual grid adjustments
Manual grid adjustments
Feature Extraction for CytoGenomics
For most images, grids are adjusted adequately if you follow the steps provided by the Help Wizard. However, if the placement of the grid is not adequate after performing the steps of the Help Wizard, you can correct the grid placement using the following procedures.
To rotate the position of the main grid
To move or rotate the main grid
another way, see To move or
rotate the main grid with Grid
Adjustment pane" on page 142.
1 Make sure that the color log scale has been turned on to view the corner spots on the main grid more easily.
2 Click the Adjust Main Grid button on the toolbar.
3 Move the mouse to a corner of the main grid in the lower Corner View.
The rotate cursor appears.
4 Drag the rotate cursor to rotate and optimize the position of the grid on the four corners of the microarray image.
Drag rotate cursor.
5.2 User Guide
3 Creating Grid Files To move or rotate the main grid with Grid Adjustment pane
142
Figure 38 Final position of main grid on microarray
To move or rotate the main grid with Grid Adjustment pane
When the Main Grid View appears, the Grid Adjustment tab is the default selection. You can move or rotate the main grid by entering values for fields in the Grid Adjustment tab.
To learn all the ways that you can
move grids and spot centroids, see
Shortcuts to help you work with
grids and find spots" on page 158.
1 Click the Grid Adjustment tab, if necessary.
Figure 39 Grid Adjustment pane for Main Grid View
2 Click the Adjust Main Grid button on the toolbar.
3 Enter a value for any field that is available to you.
Array Label
Top Left Corner
Rotation
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To rotate the subgrid
To rotate the subgrid
Feature Extraction for CytoGenomics
If moving the position of the subgrid does not work, try rotating the subgrid.
1 Click to select the Adjust Subgrid button on the toolbar.
2 Click to deselect the Select Corners and Automatically Fit Grid button on the toolbar (if it is turned on).
3 Click on a subgrid in the upper Main Grid View.
4 In the lower subgrid Corner View, move the mouse to a corner of the subgrid until the rotate cursor appears.
5 Drag the rotate cursor on the subgrid Corner View to optimize the position of the grid on the four corners of that portion of the microarray image.
Figure 40 Subgrid with rotate cursor
Rotate Cursor
5.2 User Guide 143
3 Creating Grid Files To skew the subgrid corner
To skew the subgrid corner
144
If neither of the above methods work, try skewing the subgrid corner.
1 Click the Skew Subgrid button, .
2 Move the mouse to a corner of the subgrid Corner View.
The skew cursor appears.
3 Drag the skew cursor on the subgrid Corner View to optimize the position of the grid on the four corners of that portion of the microarray image.
Figure 41 Subgrid with skew cursor
Skew Cursor
To position subgrids with Grid Adjustment pane
When the Adjust Subgrid button is selected , the Grid Adjustment tab is the default selection. You can position subgrids by entering values for fields in the Grid Adjustment tab.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To position subgrids with Grid Adjustment pane
To learn all the ways that you can
move grids and spot centroids, see
Shortcuts to help you work with
grids and find spots" on page 158.
Feature Extraction for CytoGenomics
1 Click the Grid Adjustment tab, if necessary.
Figure 42 Grid Adjustment pane for subgrids
2 Enter a value for any field that is available to you.
Col and Row Numbers
Click the down arrows for Col and Row to select the column and row numbers for the subgrid you want to position.
Corner of Subgrid
Rotation
Skew
5.2 User Guide 145
3 Creating Grid Files Saving a grid file
Saving a grid file
146
Whenever you have made manual adjustments to the grid or spacing geometry, you must save the grid file in order to keep the changes you have made. There is only one grid file for any given image.
1 Click the Save Grid button on the main toolbar.
2 The grid is saved to a ImageName_grid.csv file where ImageName is the name of the image associated with the grid, and the directory containing the grid file is the same as the directory of the original image. For non- Agilent images, a features file is saved at the same time, with the name ImageName_feat.csv.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Calculating spot size and spot centroids
Calculating spot size and spot centroids
This option is available only for
non-Agilent images.
Feature Extraction for CytoGenomics
This is the same algorithm used for calculating spot size and finding spot centroids by the software during an automatic extraction. For third- party arrays, running this will show you how well the algorithm will find the spots on the array and let you perform corrections, if required (see Locating spot centroids manually" on page 148).
1 Click the Adjust Spot button to enable spot mode.
2 Click the Calculate Spot Size and Centroids button to let you see the difference between the nominal spot centroid, positioned from the grid (+), and the estimated spot centroid based on the spot size (X).
After you have calculated the spot size and spot centroids, you can make adjustments manually. See Locating spot centroids manually" on page 148. When you move a grid or subgrid after calculating the spot size and spot centroids, the found centroids for the moved grid or subgrid are deleted.
5.2 User Guide 147
3 Creating Grid Files Locating spot centroids manually
Locating spot centroids manually
If you want to save the
manually-placed spots, you must
save the grid file.
148
Once you have calculated the spot sizes and centroids (see Calculating spot size and spot centroids" on page 147) you can manually adjust those. In this section you learn how to move a nominal spot centroid, placed on a spot by the subgrid. When you move the nominal spot centroid (+), it becomes an estimated spot centroid (X). Once the spot centroid is estimated, the software uses this estimated value for Feature Extraction.
You cannot move spot centroids for
Agilent images. This function
works only for non-Agilent images.
If a spot centroid has been manually moved, the result called IsManualFlag is set to true. See the Feature Extraction for CytoGenomics Reference Guide for the result.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To find a spot to adjust
To find a spot to adjust
Feature Extraction for CytoGenomics
1 Click the Adjust Spot button, ,to bring up the Main Spot View and the Zoomed Spot View.
Figure 43 Adjust spot view
Main View
Zoomed Spot View
Preview Spot Centroids button
Spot Navigator (square box)
Spot Navigator button
Spot Location Adjustment Pane
Adjust Spot button
The calculated spot centroids
appear when you enter Adjust Spot
mode.
2 Select Color > Use Log Color Scale if this has not already been selected.
3 Click a spot on the Main Spot View or on the Zoomed Spot View.
Or
5.2 User Guide
150
3 Creating Grid Files To find a spot to adjust
Click the Spot Navigator button, ,to bring up the Grid Spot Navigator dialog box.
4 Click the down arrow in the Find by list, and select the number or name of the spot to navigate to.
5 In the Query field, type the feature number or probe name that you want to find.
Case is important.
6 Click Find.
If only one feature in the microarray matches the query, the software moves the spot square (Spot Navigator) to that feature.
If more than one feature matches, a list of features is displayed in the bottom box.
7 Select a feature from the list, and click Go to.
The software moves the Spot Navigator to that feature.
8 Click Close.
The software positions the Spot Navigator (square box) in the center of the Zoomed Spot View.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To position the spot centroid
To position the spot centroid
Feature Extraction for CytoGenomics
Before you position the Spot Navigator to find the centroid, the position of the nominal spot centroid could look like this:
Nominal spot centroid (+) to be moved
Move the Spot Navigator square containing the nominal spot centroid position to the assumed center of the spot.
The resulting position might look like this:
New estimated spot centroid (X) position;
These images represent non- Agilent microarrays. You will more likely need to position the spot centroid for non- Agilent microarrays.
When this altered grid file is used in Feature Extraction, the default spot finding algorithm will not attempt to move spots that have been manually located in the grid file.
5.2 Us
3 Creating Grid Files To ignore spots
To ignore spots
152
1 Position the Spot Navigator where you want to ignore a spot.
2 In the Grid Adjustment pane, click the field next to Ignore and then select True.
The software will not use this spot
for any step in Feature Extraction.
To undo or redo spot position movements
Click the Undo button, , to undo an action.
Click the Redo button, , to redo an action.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 To position spot centroids with the Grid Adjustment pane
To position spot centroids with the Grid Adjustment pane
Feature Extraction for CytoGenomics
When the Spot View appears, the Grid Adjustment tab is the default selection. You can position spot centroids by entering values for fields in the Grid Adjustment tab.
1 Click the Grid Adjustment tab, if necessary.
To learn all the ways that you can
move grids and spot centroids, see
Shortcuts to help you work with
grids and find spots" on page 158.
Figure 44 Spot Location Adjustment pane
2 Enter a value into any field that is available to you.
3 Press Enter.
Col and Row Numbers
Click the down arrows for Col and Row to select the column and row numbers for the spot centroid you want to position.
Center
SpotDiam
5.2 User Guide 153
3 Creating Grid Files To position spot centroids with the Grid Adjustment pane
ProbeName
154
Name of probe associated with selected spot; found in gene list
GeneName
Ignore
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Refitting a grid
Refitting a grid
Feature Extraction for CytoGenomics
You may want to refit a grid under the circumstances mentioned in the table below. Alternate solutions to the problems are also described:
Table 4 Conditions for refitting grids
Grid Issue Things to try before refitting the grid
Main grid is not properly placed. Adjust the placement of the main grid. See
Step 3. Adjust the position of the main
grid" on page 137.
Subgrids are not correctly spaced. Adjust the spacing between subgrids.
Assumption is that the subgrids are evenly
spaced. See Step 2. Get nominal subgrid
spacing manually" on page 136 and Step
4. Adjust the subgrid" on page 138.
Spot spacing appears off. Adjust the spacing between the spots.
Step 1. Get nominal spot spacing
manually" on page 134.
Average spot size is not correct. Adjust the spot diameter. This diameter is
used by the Calculate Spot Size and
Centroids algorithm and the WholeSpot
algorithm. See Calculating spot size and
spot centroids" on page 147 and
Locating spot centroids manually" on
page 148.
When you refit a grid, any changes you made during manual gridding, if
To refit a grid, follow these steps.
To learn about Geometry settings,
see Geometry information" on
page 129.
1 Select Edit > Refit Grid.
The Geometry Information dialog box appears.
5.2 User Guide 155
156
3 Creating Grid Files Refitting a grid
Click Reset to re-enter the original
number before you changed it.
Figure 45 Geometry Information dialog box to refit Agilent grid
2 Change entries, and click Auto Fit or Manual Fit. A message will appear reminding you that refitting the grid will cause you to lose any changes you made that have not been saved in the grid file.
3 Click Yes to continue, or No to return to the Geometry Information dialog.
4 Once the grid has been refit, save the grid file to keep the changes made by the refitting procedure.
See Saving a grid file" on page 146.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Changing default settings for Grid Editing
Changing default settings for Grid Editing
Feature Extraction for CytoGenomics
Before you begin to set up a grid, you can change the default settings that appear in the Grid Definition and Grid Adjustment panels on the main window when the grid mode is turned on.
1 Select Tools > Preferences.
2 Click the Grid Editing folder.
Figure 46 Preference SettingsGrid Editing selected
Default View Zoom setting
Click the cell next to an image window, then click the down arrow to select the zoom setting for each of the image windows listed.
5.2 User Guide 157
3 Creating Grid Files Shortcuts to help you work with grids and find spots
Shortcuts to help you work with grids and find spots
158
There are several ways that you can select and move grids and spots. These are presented in Table 5. In addition, there are a number of Ctrl and Alt key operations that help you work with grids. These are presented in Table 6.
Table 5 Grid Actions
Location of actions
If you want to do this:
Do this on the Grid Adjustment dialog box:
Or, do this with the keyboard:
Or, do this with the mouse:
Main Grid Set array label Edit value, then press Enter. NA NA
Move grid Edit value, then press Enter. Hold down the Shift key
and select an arrow key.
Click the left mouse button
and drag the cursor across
the full window. Or, click the
left mouse button in the
corner window.
Rotate grid Edit value, then press Enter. NA Drag on corner in corner
window.
Subgrids Select subgrid* Select column and row
number from a choice list.
Use arrow keys or Page
Up/Page Down (requires
two rows or more) or
Home/End
Left-click mouse in full view
or corner view.
Move subgrid Edit location, then press Enter. Hold down the Shift key
and select an arrow key.
Drag the cursor across the
full window. Or, click the left
mouse button on the top left
spot of the subgrid in the
corner window.
Rotate subgrid Edit value, then press Enter. NA Drag on corner in corner
window. Or, turn on
automatic skew and rotate
and then click the left mouse
button on the upper left,
upper right, and lower left
spot of the subgrid in the
corner window.
Feature Extraction for CytoGenomics 5.2 User Guide
Creating Grid Files 3 Shortcuts to help you work with grids and find spots
Skew subgrid Edit value, then press Enter. NA Click Skew Subgrid button,
then drag on corner in corner
window. Or, turn on
automatic skew and rotate
and then click the left mouse
button on the upper left,
upper right, and lower left
spot of the subgrid in the
corner window.
Spots Select spot* Select column and row
number from a choice list.
Use arrow keys or Page
Up/Page Down or
Home/End
Left-click mouse in full view
or corner view.
Move spot
centroid
Edit location, then press Enter. Hold down the Shift key
and select an arrow key.
Drag Spot Locator to position
spot centroid.
Flag spot Mark the Ignore this spot check box, then press Enter.
NA Double-click the right mouse
button to toggle this flag.
* These are the only grid actions that you cannot undo, redo or reset.
Table 5 Grid Actions (continued)
Location of actions
If you want to do this:
Do this on the Grid Adjustment dialog box:
Or, do this with the keyboard:
Or, do this with the mouse:
Feature Extraction for CytoGenomics
Table 6 Hot Key Actions for Grid Mode
If you want to do this: Press this hot key:
Turn Grid Mode On/Off Alt-G
Select for Main Grid adjustment Alt-M
Select for Subgrid adjustment Alt-S
Select for Spot adjustment Alt-P
Select to ignore a spot Ctrl-G
Undo Ctrl-Z
Undo All Ctrl-A
Redo Ctrl-Y
5.2 User Guide 159
160
3 Creating Grid Files Shortcuts to help you work with grids and find spots
Redo All Ctrl-D
Refit grid Ctrl-T
Table 6 Hot Key Actions for Grid Mode (continued)
If you want to do this: Press this hot key:
Feature Extraction for CytoGenomics 5.2 User Guide
Agilent CytoGenomics 5.2 Agilent Feature Extraction for CytoGenomics User Guide
4 Changing Protocol Settings
Changing protocol settings 162
Creating a new protocol from an existing protocol 163
Changing Protocol Step 1: Place Grid 172
Changing Protocol Step 2: Optimize Grid Fit 178
Changing Protocol Step 3: Find Spots 182
Changing Protocol Step 4: Flag Outliers 191
Changing Protocol Step 5: Compute Bkgd, Bias and Error 199
Changing Protocol Step 6: Correct Dye Biases 216
Changing Protocol Step 7: Compute Ratios 222Changing Protocol Step 8:
Calculate Metrics 227
Changing Protocol Step 9: Generate Results 230
This chapter gives you background information on changing settings for the protocols used to perform Feature Extraction. The Feature Extraction protocols contain the parameter values for the algorithms that you specify for each step of the Feature Extraction process.
See Chapter 1 in the Agilent Feature Extraction for CytoGenomics Reference Guide for a listing of the default settings and parameter values for the protocols shipped with the software.
161Agilent Technologies
4 Changing Protocol Settings Changing protocol settings
Changing protocol settings
Protocol templates
162
A protocol template for CGH extractions is provided with Agilent Feature Extraction for CytoGenomics. The name of protocol templates follow the format:
Agilent microarray type_software version_date
For a complete list of the default protocol settings, see the Agilent Feature Extraction for CytoGenomics Reference Guide.
Both the protocol parameters and default values may change over time. To ensure you have the current protocol, from the Feature Extraction main window, click Help > Feature Extraction on the Web > Feature Extraction Protocols.
To create a new protocol from a protocol template
See Chapter 5, How Algorithms
Calculate Results in the Feature
Extraction for CytoGenomics
Reference Guide for detailed
information on how the protocol
steps and algorithms work.
Each protocol contains protocol steps that represent Feature Extraction algorithms. In the FE Protocol Editor, you make changes to the parameter values for each of the steps.
After you open the FE Protocol Editor for a specific protocol, you can change settings, but you must then save the protocol to a new name. The process is as follows:
1 To open a protocol.
2 To view or change the protocol properties.
3 To change the protocol steps to run.
4 To change the settings for each protocol step.
5 To save a protocol with a new name.
The following sections present instructions for creating protocols from templates and tasks for changing settings.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 Creating a new protocol from an existing protocol
Creating a new protocol from an existing protocol
Feature Extraction for CytoGenomics
To create a protocol for a specific type of microarray, you must use an existing protocol for the same type of microarray as a template.
For example, if you want to create a new protocol to run a gene expression 2- color microarray, start by editing an Agilent- created protocol for gene expression 2- color microarrays.
You can track what the original Agilent- created protocol is by looking at the Derived From field of the general section of the new protocol. See To change the protocol steps to run" on page 166.
CAUTION Existing protocols can be used as templates for new protocols to
provide both visible and hidden settings whose values are specific to
the type of microarray. Although you can change the visible settings of
any two protocols of different types to appear identical, you cannot change the hidden settings that distinguish these protocols from one
another.
5.2 User Guide 163
4 Changing Protocol Settings Creating a new protocol from an existing protocol
To open a protocol
164
You can open the FE Protocol Editor in one of three ways:
In the Protocol Browser double- click the protocol that you intend to edit, or right- click the protocol and select Properties.
In Project Explorer, either double- click the protocol assigned to an extraction set, or right- click the protocol and select Properties.
Click the Extraction Configuration tab, and in the Protocol column double- click the protocol that you intend to edit.
Regardless of the protocol, the first screen that appears is by default the screen that was last displayed before a protocol was closed or saved.
Figure 47 FE Protocol Editor Protocol Steps
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 Creating a new protocol from an existing protocol
To view or change the protocol properties
Feature Extraction for CytoGenomics
The General section of the Protocol Properties page lists fields to identify the protocol and fields to let you remove the protocol or make the protocol read- only.
If necessary, in the FE Protocol Editor click the Protocol Properties button to see the General section.
Figure 48 Protocol Editor Protocol Properties tab
Date and Time
Protocol Version
Derived From
Metricset
5.2 User Guide 165
4 Changing Protocol Settings To change the protocol steps to run
Protocol Type
166
Corresponds to type of microarray for which the protocol is to be used for Feature Extraction: 2- color gene expression, 1- color gene expression, CGH, ChIP, or miRNA data.
Protocol Removable
Describes if the protocol is removable from the database. User- created protocols are removable. Agilent- created protocols are not removable.
Description
Permanent Read Only
True indicates that the protocol cannot be edited. Any changes made to the protocol must be saved with a different name.
False indicates that the protocol can be edited. Any changes made to the protocol can be saved with the existing name.
To change the protocol steps to run
If there is no protocol on your system with the steps you want Feature Extraction to use, you can open a protocol of the appropriate type and change the protocol steps.
1 If necessary, in the FE Protocol Editor click the Protocol Properties button to see a list of the protocol steps on the right.
2 Mark each check box to enable a step that you want to run. Clear each check box to disable a step that you do not want to run.
Refer to Figure 49 to see the FE Protocol Editor.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change the protocol steps to run
Feature Extraction for CytoGenomics
Protocol steps for CGH images
Below is a description of each of the protocol steps for CGH, images, as seen in Figure 49, in the order that the software runs them.
Place Grid
For any microarray loaded with a grid file that you saved during Grid Mode (i.e. interactive gridding), automatically calculates centroid positions. Any spots that were manually adjusted are left unchanged.
For Agilent microarrays, the software performs a background peak shift that helps find peaks of features that are close to the background. See the Feature Extraction for Cyto Reference Guide for details.
For any microarray, user can select to use regions near to the center of packs for slope and skew calculations. This option is of use in cases where pack edges have dim spots and are failing to grid. When this is selected, the user can select to have the software to perform one extra step of correlation following the projection data analysis to get the origin.
Optimize Grid Fit
Find Spots
From the located spots, the features are defined with either a CookieCutter method or a WholeSpot method.
The local background is also defined with a radius method.
Outlier pixels (too low or high intensity) are then removed from the pixel populations that defined the features and backgrounds.
5.2 User Guide 167
168
4 Changing Protocol Settings To change the protocol steps to run
Statistics, such as the mean, median and standard deviation (SD), are calculated on the remaining inlier pixels within the feature and background.
Features with a large number of saturated pixels are flagged.
The protocol determines whether the mean or median signals are used for further analysis.
Flag Outliers
Features that have replicates of the same probe sequence and background regions are flagged as population outliers if their signal distributions are outside the set range.
Compute Bkgd, Bias, and Error
If background subtraction is selected, background signal is subtracted from raw signal for each feature using a local or global background method.
A statistical test is performed to assess if the feature signal is significantly different from the background signal. This test uses either the error model or pixel statistics, as indicated in the protocol choice Significance.
Raw signals or background- subtracted signals can be corrected for systematic gradients, that affect low signals especially, by using additive detrending (i.e. spatial detrending).
The error is calculated at this point, using the choice between the Universal error model or the larger of the error estimates (i.e. most conservative) from the Universal error model and the propagated error.
A multiplicative detrending algorithm is also used to correct for gradients proportional to the signal.
Surrogates, based upon background noise, can be used as processed signals for features that have low- intensities, near the background noise level.
Correct Dye Biases
The method for how a dye normalization gene list is selected is set.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change the protocol steps to run
Feature Extraction for CytoGenomics
Differences in the red and green channel signals caused by different efficiencies in labeling, emission and detection are estimated and corrected.
Features to be used for measuring dye bias are selected based on a probe selection method chosen.
A dye normalization curve fit method is selected and used to fit the data for measuring dye bias. (SNP probes are not included in dye normalization calculations.)
Compute Ratios
Calculate Metrics
In this step, the Stats table is generated and QC metrics are calculated for the QC Report.
You must leave this option turned on to generate a complete Stats table and QC report. Some values will be missing if this option is turned off.
The type of protocol you select determines the type of QC report that is generated.
For example, a CGH protocol generates a CGH QC Report, and a 1- color protocol with spike- ins selected generates a 1- color QC Report with spike- ins.
Generate Results
Results selected for the project are generated.
In the Protocol Editor you can select to generate one file for the three tables (FEPARAMS, STATS, FEATURES) of text results or three separate files. You can set the compression for the jpeg file.
5.2 User Guide 169
4 Changing Protocol Settings To change the settings for each protocol step
To change the settings for each protocol step
170
After you change the protocol steps that you want to run, you may want to change the default settings and parameter values for a specific protocol step.
1 Click the Protocol Steps tab to see a list of the protocol steps, or Feature Extraction algorithms, on the left.
2 Click the protocol step button whose settings you intend to change.
Figure 49 FE Protocol Editor with Protocol Steps on left
See the remaining sections in this chapter to learn how and why you change parameters for each protocol step.
To save a protocol with a new name
After you make any change to a protocol, you must save the protocol to a new name.
1 Make the changes you intend.
2 Click Save As.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To save a protocol with a new name
Feature Extraction for CytoGenomics
Figure 50 Add new protocol dialog box
3 Enter a Protocol name.
4 Click OK.
5.2 User Guide 171
4 Changing Protocol Settings Changing Protocol Step 1: Place Grid
Changing Protocol Step 1: Place Grid
172
The Place Grid algorithm initiates Feature Extraction by placing a grid on the microarray image. The grid comes from either the grid template or the grid file associated with the image file in the project.
To access the settings to place a grid
Click the Place Grid tab in the FE Protocol Editor, if not already there.
Figure 51 Protocol Editor Place Grid step
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To access the settings to place a grid
To select the microarray format
Feature Extraction for CytoGenomics
This topic describes how to select an array format if needed. You typically do not need to do this step, as the software automatically determines which array format and scan type is used.
Once you change to a specific array format, the protocol loses its ability to be general and will only work for that specific format.
1 Click the cell to the right of Array Format.
2 Click the down arrow, and select a microarray format from the list.
Figure 52 Place Grid Array Format list
The Placement Method option now appears. See Figure 56 on page 180.
5.2 User Guide 173
174
4 Changing Protocol Settings To access the settings to place a grid
Figure 53 Place Grid 30-micron multi pack Array Format selected,
Placement Method
After you select the format, you can select a method for placing the grid.
Protocols can automatically detect the array format (65- micron feature size or 30- micron feature size). The protocol automatically makes different parameter choices depending on the format and type. These choices are transparent to you and are primarily in the image processing (Place Grid and Find Spots).
With this feature, you do not need different protocols for different array formats. These protocols contain the correct parameter choices for each and use the set that matches the format.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select a method for placing the grid
Feature Extraction for CytoGenomics
Information about which parameters are affected and which parameters are chosen for each format type can be found beneath any option with a choice automatically determine. If you believe an incorrect automatic choice was made, you can select a specific format. However, this is not a normal or necessary step.
To select a method for placing the grid
1 Click the cell next to Placement Method, and click the down arrow.
2 Select one of the two options listed below.
Allow Some Distortion
Allows some variance in the feature spacing, subgrid spacing and the skew. All of the protocol templates have this default setting.
Place and Rotate Only
Only allows rotation of the grid and no changes to feature spacing, subgrid spacing or the skew.
To select to use enhanced gridding
As of version 5.0, Feature Extraction for CytoGenomics offers an enhanced gridding algorithm used in the preloaded protocols that was not available in protocols that were created prior to version 5.0. These enhancements were designed to improve automatic gridding in array images of lower quality, including those with non- linear features. Array images that may have previously received a failed grid error and require manual gridding can now be processed successfully using the enhanced algorithm.
Importantly, use of the enhanced algorithm may yield changes in some of the QC metrics results and aberration calls compared to the previous algorithm. Validation is recommended.
5.2 User Guide 175
176
4 Changing Protocol Settings To select to enable background peak shifting
If you are editing an imported protocol of any type, the enhanced gridding setting is off by default.
1 Click the cell next to Use Enhanced Gridding, and click the down arrow.
2 Select True or False.
When set to True, the enhanced gridding algorithm that was added in Feature Extraction for CytoGenomics 5.0 is used in the protocol.
When set to False, the enhanced gridding algorithm that was added in Feature Extraction for CytoGenomics 5.0 is NOT used in the protocol. The previous version of the gridding algorithm is used instead.
To select to enable background peak shifting
1 Click the cell next to Enable background peak shifting? and click the down arrow.
2 Select True or False.
If you select True, the background pixel value is obtained using the histogram of the image. All the pixels in the image that belong to background are reassigned a value of zero. Note that this assignment is temporary and does not change the actual image data for subsequent steps.
To select to use central part of pack for slope and skew calculations
This option lets you choose the region to use for slope and skew calculation during subgrid placement.
1 Click the cell next to Use central part of pack for slope and skew calculations? and then click the down arrow.
2 Select True or False.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select to use the correlation method to obtain origin x of subgrids
Feature Extraction for CytoGenomics
When set to True, regions near to the center of packs are used. This option is of use particularly in cases where pack edges have dim spots and are failing to grid. (This is the default setting for 30 micron single- and multi- pack array formats.)
When set to False, the regions near to the edges of packs are used. Selecting this option uses the same parameters as Feature Extraction version 10.7/10.9 or earlier.
To select to use the correlation method to obtain origin x of subgrids
This option lets you choose the technique to use to determine X location of origin of subgrids.
1 Click the cell next to Use central part of pack for slope and skew calculations? and click the down arrow.
2 Select True.
3 Click the cell next to Use the correlation method to obtain origin x of subgrids? and then click the down arrow.
4 Select True or False.
When set to True, the software performs one extra step of correlation following the projection data analysis to get the origin. This option is of use particularly in cases where pack edges have dim spots and are failing to grid.
When set to False, the results obtained from the projection data analysis are used to estimate the origin. This is how the origin is estimated using Feature Extraction version 10.7/10.9 or earlier.
5.2 User Guide 177
4 Changing Protocol Settings Changing Protocol Step 2: Optimize Grid Fit
Changing Protocol Step 2: Optimize Grid Fit
178
The Optimize Grid Fit step is a post grid placement process to improve the grid registration. Leveraging from the spot finder algorithm, this step examines the spots in the four corners of the microarray to iteratively adjusts the grid for best fit.
To access the settings to optimize the grid fit
Click the Optimize Grid Fit tab in the FE Protocol Editor.
Figure 54 FE Protocol Editor Optimize the Grid Fit selected
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To access the settings to optimize the grid fit
To select the grid format
Feature Extraction for CytoGenomics
1 Click on the cell next to Grid Format.
2 Click the down arrow, and select from the list of grid formats.
Figure 55 Optimize Grid Fit Grid Format list
If you select either 65- micron or 30- micron feature size, the Iteratively Adjust Corners? option appears. See Figure 53 on page 174.
5.2 User Guide 179
180
4 Changing Protocol Settings To change the settings to iteratively adjust corners when grid format is selected
Figure 56 Optimize Grid Fit 30-micron Feature Size selected,
Iteratively Adjust Corners
To change the settings to iteratively adjust corners when grid format is selected
1 Click the cell next to Iteratively Adjust Corners?.
2 Either select True or False.
3 If you select True, you can change any of the default parameters that you need to.
When set to True, the Interactively Adjust Corners method runs the spot finder algorithm in the corners to see how well the grid is placed on the microarray and iteratively adjusts the grid based upon the results.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change the settings to iteratively adjust corners when grid format is selected
Adjustment Threshold
Feature Extraction for CytoGenomics
The grid is adjusted based upon the difference between the grid position and the spot position if the absolute average difference is greater than this value.
Maximum Number of Iterations
This is the maximum number of times the spot finder algorithm runs to adjust the grid for better grid fitting in the corner spots.
Found Spot Threshold
Number of Corner Features Side Dimension?
This specifies the number of features (i.e. spots) on each side of the square in each corner of the microarray to be used to calculate the average difference.
The default number is 20 for the 30- and 65- micron feature size microarrays. This means each corner of the microarray has a square area of 400 (i.e. 20 x 20) features that are calculated.
5.2 User Guide 181
4 Changing Protocol Settings Changing Protocol Step 3: Find Spots
Changing Protocol Step 3: Find Spots
See Chapter 1, Default Protocol
Settings, of the Feature Extraction
for Cyto Reference Guide for a
setting comparison between the
various microarray formats.
182
After Feature Extraction places the grid, it is ready to locate the spot centers and define the features and local background regions for each spot. The options available and default values for each setting depend on the microarray format selected in Step 1: Place Grid.
To access the settings to find spots when spot format is automatically determined
Click the Find Spots tab in the FE Protocol Editor.
Figure 57 FE Protocol Editor Find Spots selected
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select the statistical method to reject pixel outliers
A spot is a circular or amorphous
area on the microarray containing a
hybridized sample.
A feature is defined as the pixels in
either the center portion of a spot
or in the whole spot, depending on
the method you choose to define
the feature.
Feature Extraction for CytoGenomics
When the spot format is automatically determined (default setting), these are the only settings available for change:
Select the statistical method to reject outlier pixels from features and local background before Feature Extraction calculates feature and background statistics.
Select whether to use mean or median for data analysis
To select the statistical method to reject pixel outliers
1 Click the cell next to Pixel Outlier Rejection Method, and click the down arrow.
2 Select one of these three choices:
Standard Deviation
This calculation rejects outliers outside of a specified number of standard deviations from the mean of the intensity for all pixels. If selected, you can enter the number of standard deviations (Feature SD and Background SD).
Inter Quartile Region
This calculation rejects outliers based on the Interquartile Range (IQR). See the Feature Extraction for Cyto Reference Guide for how this calculation works. If selected, you can enter the IQR factor for both the feature and background (RejectIQRFeat and RejectIQRBG).
None
Feature Extraction rejects outlier pixels after calculating statistics on the intensities of all the pixels of a feature or local background region for each color. If a pixel is rejected in one color, it is rejected in both colors.
5.2 User Guide 183
4 Changing Protocol Settings To select whether to use mean or median for data analysis
To select whether to use mean or median for data analysis
184
1 Click the cell next to Statistical Method for Spot Value from Pixels, and click the down arrow.
2 Select one of these choices to be used in the next step of data analysis:
Mean
Median
During the background correction stage the background can be subtracted from either the mean signal or the median signal. This option lets you decide which signal type to use for downstream analysis.
To enable the Enhance Spot Finding option
1 Click the cell next to Use Enhanced SpotFinding, and click the down arrow.
2 Either select True or False.
If you select True, then the enhanced spot finding method for is used during the spot finding step of the protocol. This method allows for more accurate identification of the center of each spot and of the pixels that comprise each spot. The result is that fewer features are called as non- uniform.
If you select False, then the protocol does not use the enhanced spot finding method. The Use Enhanced SpotFinding setting is disabled when the Use Enhanced Gridding setting (in the Place Grid menu) is set to False. See To select to use enhanced gridding" on page 175.
Note that enabling the Use Enhanced SpotFinding setting can slightly increase the array processing time.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To enable the Enhance Spot Finding option
To access the settings to find spots when spot format is selected
Feature Extraction for CytoGenomics
1 Click the cell to the right of the Spot Format.
2 Click the down arrow, and select from the list of spot formats.
Figure 58 Find Spots 30-micron Spot Format selected
In this protocol step you can also change the values for 3 additional sets of parameters associated with Feature Extraction processes to find spots once you have selected a spot format:
Change values to find and size spots.
Select a spot statistics method to define features.
Select to autoestimate the local radius.
5.2 User Guide 185
4 Changing Protocol Settings To change values to find and size the spots.
To change values to find and size the spots.
186
1 Click the cell next to the names of the following parameters.
2 Either select True or False, or enter a value, whichever is applicable.
Use the Nominal Diameter from the Grid Template
If you select True, the nominal diameter of each spot is obtained from the grid template. If you select False, the nominal diameter that is the starting spot size assumption for the Spot Finding algorithm is obtained from the grid placement algorithm.
Spot Deviation Limit
Enter a value that defines the distance the actual centroid may move relative to its nominal grid position (stated as a multiple of nominal radius). This parameter appears in the FEPARAMS table as SpotAnalysis_kmean_cen_reject.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change values to find and size the spots.
To select a spot statistics method to define features
Feature Extraction for CytoGenomics
1 Select either Use Cookie or Use Whole Spot.
Cookie Method
The Cookie method is usually used
for Agilent microarray spots
because of their uniformity.
Figure 59 Feature and background definitions Cookie method
Local backgroundring area between blue
dashed circle and red dashed circle
Exclusion zonering area between feature
and blue dashed circle outside of spot. The
pixels in this zone are not included in feature
or local background statistics.
Edge of spotblack circle
Cookie or featuregreen circle in middle
Whole Spot Method
This method uses a detection technique based on a noise model to define the feature and exclusion zone. This method performs the spot size calculation first and defines the feature as the whole spot from the x and y diameters.
Figure 60 Feature and background definition Whole Spot method
Local backgroundarea between blue
dashed circle and red dashed circle
Exclusion zonearea between edge of
feature (spot) and blue dashed circle outside
of spot
Edge of spotblack contour
Featuregreen area; entire spot
5.2 User Guide
188
4 Changing Protocol Settings To change values to find and size the spots.
2 If you selected Use Cookie, change the Cookie Percentage and/or the Exclusion Zone Percentage, if necessary.
If you selected Use Whole Spot, change the Exclusion Zone Percentage, if necessary.
Cookie Percentage
Exclusion Zone Percentage
Percentage of the nominal radius that is the exclusion zone radius.
Figure 61 Definitions of different radii
Exclusion zone radius from centroid of
spot to edge of blue-dashed line.
Cookie radius from center of
spot to edge of green circle
Nominal radius from spot centroid
to edge of spot
Local background radius from spot centroid to edge
of red circle
See the Feature Extraction for Cyto
Reference Guide for more
information.
The radii depicted in Figure 61, Definitions of different radii, on page 188 are calculated differently for the two methods.
Feature
Changing Protocol Settings 4 To change values to find and size the spots.
To select to autoestimate the local radius to define local background
Feature Extraction for CytoGenomics
This radius method estimates background that is local to a feature of interest.
1 Click the cell next to Autoestimate the local radius.
2 Select True or False.
For non-Agilent microarrays when
there is a big difference in spot
sizes, change this setting to False
and make sure that the number you
enter is big enough to
accommodate the largest spot on
the microarray.
3 If you select False, enter a larger radius if you want to include more background pixels or a smaller one if you want to include fewer.
If you select True, the software automatically estimates the radius to use to estimate the local background.
This approach eliminates any dependence of the local background computation on distance between spot- centroids along both axes. This ensures elimination of cross- contamination between feature and background signals, especially in closely packed microarrays.
See the Feature Extraction for Cyto
Reference Guide for details on this
calculation.
The default radius produces a local background calculation around only one spot (Self- radius). If you want to include more background, you can increase the radius to include the background around the spots nearest neighbors (NN radius).
5.2 User Guide 189
4 Changing Protocol Settings Propagation of Find Spot errors to other algorithms
Final calculations for protocol step to find and measure spots
190
Final statistics
The final statistics calculated for all colors of each feature and local background, based on the remaining inlier pixels, are the average, standard deviation, median, IQR, and number of pixels. In addition, the pixel correlation between the red and green channels is calculated.
Pixel saturation
Saturation is defined as the
maximum allowed intensity value
for a pixel, Its value depends on the
make and model of the scanner.
Pixel saturation is also tested during calculation of the feature statistics. For the Agilent Microarray Scanner system, saturation is defined as an intensity value of 65535 minus the dark offset from the scanner. If more than 50% of the pixels remaining after outlier rejection are saturated, Feature Extraction sets a saturation flag.
With XDR extractions, the saturation limit could be as much as 20x higher than 65535. The array will saturate before it gets to 20x of 65535. The calculated saturation value based upon the XDR fit could be as high as 1,310,000 counts, but no biological signals are that high.
20- bit scans also have a high saturation value, one close to 1,048,576 counts.
Propagation of Find Spot errors to other algorithms
The measurement errors from the calculation of these statistics are propagated to the next steps in Feature Extraction according to standard error propagation techniques. For example, the errors for the signal average for the feature and local background regions are propagated to the background subtraction algorithm.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 Changing Protocol Step 4: Flag Outliers
Changing Protocol Step 4: Flag Outliers
Feature Extraction for CytoGenomics
Not every feature is created, hybridized and scanned perfectly. Some backgrounds contain artifacts. Therefore, you can flag those features or local backgrounds that may represent misleading or erroneous data.
Although the grid tool of the Feature Extraction software lets you manually examine scanned microarray images and flag anomalous features, this task becomes exceedingly tedious and subjective for thousands of features on a microarray. This algorithm automatically finds all the anomalous features on the microarray.
To access the settings to flag outliers
Click the Flag Outliers tab to change settings for flagging abnormal features and backgrounds.
5.2 User Guide 191
192
4 Changing Protocol Settings To access the settings to flag outliers
Figure 62 FE Protocol Editor Flag Outliers selected
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change settings to flag population outliers
Feature Extraction for CytoGenomics
You can change two types of settings for this algorithm:
Change settings to flag population outliers.
With this option you can flag features and local background regions that have intensities beyond the confidence limit for the distribution of the intensities for the replicate features and local background regions on the microarray.
Change settings to flag non- uniform features and background region (outliers).
To change settings to flag population outliers
A type of outlier, called a population outlier, can be detected if there are enough replicates to perform population statistics on the signal intensities of the features or the signal intensities of the background regions.
For example, there are a number of control features on a typical Agilent microarray (typically, over 100 features) and thousands of local background regions. Out of this population, outliers can be rejected using box plot analysis.
1 Click the cell next to Compute Population Outliers, and click the downarrow.
2 Select True or False.
3 If you select True, change any of the default parameters that you need to.
The Flag Outliers tab contains values for the minimum population size to be considered for population outlier flagging, as well as a cutoff range. If a feature or local background region signal lies outside of this range, Feature Extraction flags that feature or local background region as a population outlier.
Minimum population
This is the minimum number of replicates needed to perform the population outlier analysis. Although 10 is the default number of replicates, you can enter as low as 5 replicates.
5.2 User Guide 193
194
4 Changing Protocol Settings To change settings to flag non-uniform outliers
To take advantage of the replicated probes in statistical calculations, Agilent uses the default threshold of 10 because its catalog microarrays have 100 probes that are replicated 10 times on the microarray. The default value for non- Agilent microarrays is 15.
IQRatio
Background IQRatio
This option uses the IQRatio formula for estimating the background region outliers.
Use Qtest for Small Populations?
When the number of replicates falls below the minimum number specified in the protocol, a Q- test can be used to estimate the number of population outliers instead of the IQRatio. See Chapter 5 of the Feature Extraction for Cyto Reference Guide to learn more about Q- test calculations.
Report Population Outliers as
Failed in MAGEML file
When this option is set to True, any feature that is flagged as a population outlier is reported as Failed in the MAGE- ML output file.
To change settings to flag non-uniform outliers
The Agilent Microarray system
includes Agilent microarrays,
label/hyb/wash protocols and
reagents, scanner and Feature
Extraction algorithms.
The non- uniformity outlier algorithm flags anomalous features or local backgrounds based upon statistically significant deviations from a noise model that makes use of the known noise characteristics of the Agilent Microarray system.
You do not typically need to do this step, as the software automatically determines the scan type and sets the parameters appropriately. To view or change the parameters
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change settings to flag non-uniform outliers
Feature Extraction for CytoGenomics
that are automatically set, select a specific scan type. However, if you change the parameters, the protocol is no longer general and can only be used with that scan type.
1 Click the cell next to Compute NonUniform Outliers, and click the downarrow.
2 Select True or False.
True is the default setting for all Agilent- created protocols. If True is set, move on to the next step.
3 Click the cell next to Scanner, and click the down arrow.
4 Select either Automatically Determine or Agilent Scanner.
The settings change depending on your selection.
To learn more about how the
algorithm calculates the terms in
the polynomial equation below, see
the Feature Extraction for Cyto
Reference Guide.
5 Click the cell next to Automatically Compute OL Polynomial Terms.
6 Select True or False.
True is default setting. A different set of parameters appears for a True or a False setting.
7 Enter the values for the parameters.
5.2 User Guide 195
196
4 Changing Protocol Settings To change settings to flag non-uniform outliers
Parameters for a True setting
Figure 63 Flag Outliers Agilent Scanner selected,
Automatically Compute OL Polynomial Terms: True
The polynomial equation for the noise model is still used, but rather than entering values for the A, B and C terms as in the False case, you enter the values for the A (%CV)^2 term and multipliers for the B and C terms in both color channels (2- color) or one channel (1- color).
(%CV)2 Term (A)
Red Poissonian Noise Term (B) Multiplier
The B term for the red channel is equal to this multiplier times the net signal at the 25% percentile of a histogram plot of number of negative control features or background vs. net signal.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change settings to flag non-uniform outliers
Red Signal Constant Term (C) Multiplier
Feature Extraction for CytoGenomics
The C term for the red channel is equal to this multiplier times the variance at the 25% percentile of a histogram plot of number of negative control features or background vs. variance.
Green Poissonian Noise Term (B) Multiplier
The B term for the green channel is equal to this multiplier times the net signal at the 25% percentile of a histogram plot of number of negative control features or background vs. net signal.
Green Signal Constant Term (C) Multiplier
The C term for the green channel is equal to this multiplier times the variance at the 25% percentile of a histogram plot of number of negative control features or background vs. variance.
CAUTION The default values for these terms have been optimized for Agilent
microarrays, with an Agilent scanner and recommended lab protocols.
You must perform optimization experiments to determine the optimum
values for your microarrays and lab protocols.
5.2 User Guide 197
198
4 Changing Protocol Settings To change settings to flag non-uniform outliers
Parameters for a False setting
Figure 64 Flag Outliers Agilent Scanner selected,
Automatically Compute OL Polynomial Terms: False
A noise model, based upon a polynomial equation, is made up of three variables whose values are shown in the Automatically Compute OL Polynomial Terms folder:
(%CV)2 Term (A)
Poissonian Noise Term (B)
This term specifies the variance due to Poisson distributed noise, which accounts for error in counting statistics.
Background Term (C)
This term specifies the variance due to signal- independent background noise (e.g., scanner and glass substrate noise).
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 Changing Protocol Step 5: Compute Bkgd, Bias and Error
Changing Protocol Step 5: Compute Bkgd, Bias and Error
Feature Extraction for CytoGenomics
At this stage of the Feature Extraction process, several systematic sources may still remain that contribute to the background signal component of the feature signal:
Scanner offset, which can vary from 20 counts to 300 counts, depending upon the version of Agilent scanner [B (20) or A (300) scanner]. The value for each microarray offset is shown in the Stats table in the fields: gDarkOffsetAverage and rDarkOffsetAverage (See the Feature Extraction for Cyto Reference Guide.)
Glass and any contaminants or non- specifically- bound fluorescent signal on the glass. Artifacts from labeling, hybridization and washing steps can contribute to this source.
Fluorescent signal that is non- specifically associated with the DNA probes themselves.
A systematic gradient across a microarray.
Background region levels can also vary depending on the dye label and color channel used.
5.2 User Guide 199
4 Changing Protocol Settings Changing Protocol Step 5: Compute Bkgd, Bias and Error
To access the settings to compute background, bias and error
200
Click the Compute Bkgd, Bias and Error tab to select a method and change settings for background subtraction.
Figure 65 FE Protocol Editor Correct Bkgd, Bias and Error
The selections for this protocol step let you decrease the contribution of the background signal to the feature signal.
To view the values used for
background subtraction depending
on the subtraction and correction
methods selected, see the Feature
Extraction for Cyto Reference
Guide.
Select the background subtraction method.
Choose to decrease the signal contribution due to an additive gradient at low signal across the microarray spatial detrend.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select the background subtraction method
Feature Extraction for CytoGenomics
Choose to adjust the signal contribution due to a systematic multiplicative gradient across the microarray multiplicative detrend. SNP probes are not included in multiplicative detrending.
Choose to globally adjust the initial background- subtracted signals.
Choose an error method to estimate the error in the calculations for the processed signal.
Change settings to assess if the background- subtracted signal is significant compared to the background.
Choose to use or not use surrogates to improve the reliability of low- signal error calculations.
To select the background subtraction method
There are several methods for subtracting the background signal from the raw signal, both local and global, available in the Correct Bkgd and Signal Biases sheet.
1 Click the cell next to Background Subtraction Method, and click the down arrow.
2 Select the background subtraction method most relevant to your microarray and processing conditions.
The default method for Agilent microarrays is No background subtraction. The default method for non- Agilent microarrays loaded with a grid file is Minimum signal (feature) on the microarray. This option makes the fewest assumptions about the microarray system. If you are analyzing non- Agilent microarrays or procedures, you have to determine the optimum default method.
No Background Subtraction
Even though no background is subtracted, you can make corrections to the signal using spatial detrend and the global background adjustment tools. The spatial detrend algorithm will correct for sources of background that are available in this protocol step, in addition to correcting for spatial gradients.
5.2 User Guide 201
4 Changing Protocol Settings To select the background subtraction method
Local background
method
202
This method subtracts the local background estimated for each spot using the radius method in the Find Spots protocol step. For example, local background for spot 300 is subtracted from the feature raw signal of spot 300.
The local background method may be a better background subtraction method than a global background subtraction method if your microarray has large regions of local backgrounds with much higher signals than the rest of the microarray.
These regions are often due to hybridization or wash processing artifacts. The local background method subtracts these local variations. This yields a better estimate of true feature signal if the local background signal is correlated with the feature signal, as determined by replicate statistics.
Global methods The Feature Extraction software supports a number of global background subtraction methods in case local background subtraction is not appropriate. With a global method, one number is calculated as background for the entire array. That number is subtracted from every feature. The number is reported in the STATS table for later queries. Global methods require that both non- uniformity outlier and population outlier methods be turned on. See the topic Changing Protocol Step 4: Flag Outliers" on page 191 for more information.
Average of All Background
Areas
This global method subtracts the average of the local background inliers estimated for all the spots using the radius method in the Find Spots protocol step. Inlier background regions have passed both the non- uniformity and population algorithms.
For example, the average background for all spots is subtracted from the feature raw signal of spot 300 and every other spot on that microarray.
Average of Negative Control
Features
If the local background is not additive and your microarray has validated negative controls, Agilent recommends using the Average of negative control features option. If the local
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select the background subtraction method
Feature Extraction for CytoGenomics
background is not additive and your microarray does not have negative controls, Agilent recommends the Minimum signal (feature) option.
Negative controls are probes that
consistently show very little target
binding across many types of
targets and have no specific target
bound to them by design.
This global method subtracts the average of the inliers of all the negative control features from each feature on the microarray. Inlier negative control features have passed both the non- uniformity and population algorithms. For example, the average of the inlier negative control features is subtracted from spot 300 and every other spot on that microarray.
Agilent provides validated negative
controls for catalog microarrays.
These probes have been shown to
reflect the non-specific signal level
that is present on probes.
If there is more than one probe sequence on the microarray annotated in the grid template or grid file as a negative control, this method analyzes the population of feature signals of each sequence to determine if there are significant differences among the different sequences at a fixed significance level of 0.01. The method then chooses the probe sequences that have the lowest mean signals and that are not significantly different from each other as the population of features to calculate negative control statistics.
Minimum signal (feature
or background)
This global method subtracts the minimum signal on a microarray from each spot, whether the signal is a feature or local background region. The feature or background region signal chosen for the minimum signal must pass a low pixel standard deviation requirement. For example, the minimum feature or background signal on the microarray is subtracted from spot 300 and every other spot on that microarray.
Minimum signal (feature)
on the array
This global method subtracts the minimum signal of a microarray feature that has passed a low pixel standard deviation requirement. This is the default setting for microarrays loaded with a grid file, but not necessarily the recommended setting. For example, the minimum feature signal on the microarray is subtracted from spot 300 and every other spot on that microarray.
5.2 User Guide 203
4 Changing Protocol Settings To choose to remove the surface trend found in the data
To choose to remove the surface trend found in the data
204
What is detrending?
To learn more about the detrending
calculations, see the Feature
Extraction for Cyto Reference
Guide.
Removing the surface trend (detrending) decreases the contribution to the background of any systematic signal gradient on the microarray. For both 2- color and 1- color data, Feature Extraction removes the additive surface trend from the data, a process called spatial detrending (background detrending). Feature Extraction also removes any domed surface trend, a process called multiplicative detrending. SNP probes are not included in multiplicative detrending calculations.
This option is the default choice with protocols for Agilent microarrays (except the miRNA protocol). The detrending is done individually for each color channel and corrected separately.
Background detrending consists of two steps: calculating a surface fit and subtracting the surface fit from the data. This process is done independently for the red and green channels after background subtraction and before the global background adjustment.
Background Spatial Detrending
For spatial detrending, Agilent separates the surface fit and subtraction calculations that must exist together to remove the surface trend found in the data.
The foreground is the portion of the
signal in a feature that is not
related to the intended signal from
the dye-labeled target
complementary to the probes on
that feature.
The surface fit calculation estimates the foreground of the data by measuring the intensity of the feature after background subtraction (or intensity of the MeanSignal of the feature if the No background subtraction option is selected) and performs a surface fit to the selected data. Even if the surface fit is not subtracted from the data (perform spatial detrending), the calculation appears in the QC Report to help evaluate your data.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To choose to remove the surface trend found in the data
Feature Extraction for CytoGenomics
The advantage of this method is that it can remove variation in foreground intensity for different regions of the microarray. By performing spatial detrending and subtracting the surface fit through this foreground, the data becomes more consistent and reproducible.
Feature Set for Surface Fit
AllFeatureTypes Select if microarray has a set of very low intensity features evenly distributed on the slide.
OnlyNegativeControlFeatures Select this if you have enough negative controls that are evenly spread around the microarray and ideally have different sequences represented default for CGHprotocols.
FeaturesInNegativeControlRange Select for low intensity features that are within 3 standard deviations of the spatial interpolated values of the negative controls.
Perform Filtering for Surface Fit
If True, allows a subset of the features into the set to be used to calculate the surface fits. You may want to do this to reduce the time needed to calculate the surface without reducing the accuracy of the fit.
If the Feature Set is AllFeatureTypes, then the filter chooses 1 to 2 percent of the dimmest features to be selected across the array. Select True for AllFeatureTypes. Select False for OnlyNegativeControlFeatures.
If the feature Set is FeaturesInNegativeControlRange, the filter chooses 1 to 2 percent of the features at random to go into the surface fitting set. Random x% rather than dimmest x% is used because the features in this set are already known to be dim.
Perform Spatial Detrending
If true, subtracts any additive trend found from the surface fit from the data.
5.2 User Guide 205
4 Changing Protocol Settings To choose to remove the surface trend found in the data
To choose to adjust the background globally (2-color)
206
To correct the errors at the low end of the signal range, Agilent lets you choose to globally adjust the background after initial background subtraction. The global adjust method was developed to be used by those customers who decide to turn off all background and detrending methods.
This adjustment helps estimate and remove background signal due to fluorescent signal that is non- specifically associated with the DNA probes and that may be under- estimated by upstream background subtraction methods. You can also select this correction if you also chose No background subtraction.
1 Click the cell next to Adjust Background Globally, and click the down arrow.
2 Select True or False.
3 If you select True, enter a pad value between 0 and 500 to force the log ratio towards zero when both red and green signals are small.
The constant supplied to the Adjust background globally algorithm pads all the feature signals by that value. This will have the effect of compressing log ratios, but will decrease the variability (standard deviation) in the log ratio among inter- and intra- array replicates. You can try small pad values, typically in the few tens, and monitor the trade- off between log ratio compression and log ratio variability. The log ratio versus signal plot is a useful preliminary tool to assess the effect of the pad value.
By setting the Adjust background globally algorithm to the same constant with multiple microarrays, the residual background can now be defined by you and is therefore more consistent across multiple microarrays and multiple experiments than if a variable residual background signal is left for each feature, if no background is subtracted.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To choose to remove the surface trend found in the data
To choose to perform multiplicative detrending
Feature Extraction for CytoGenomics
Multiplicative detrending is an algorithm designed to compensate for slight linear variations in intensity if the processing is not homogeneous across the slide, resulting in different chemical reaction concentrations, for example, between the sides and the center, a dome effect. The multiplicative gradient is removed by dividing the signal by the observed gradient. SNP probes are not included in multiplicative detrending calculations.
Make the selections described below.
Perform Multiplicative
Detrending
Choose whether to perform multiplicative detrending (can be chosen for one- or 2- color arrays).
Detrend on Replicates Only
Choose whether to detrend on all of the data or to only use those probes that have replicates. If you choose to use replicates only, the data are normalized to the respective replicate averages before going into the fitting set.
Filter Low signal probes
from Fit?
You can chose to filter out dim signals from the fit of the multiplicative trend surface. This is useful because the dimmer signals have a less obvious trend when that trend is multiplicative. You also choose how dim the signals must be in order to be removed from the set. This is stated as a multiple of the Negative Control Spread.
Perform filtering for fit
You must decide whether to use all of the data points or use local averages or medians.
5.2 User Guide 207
4 Changing Protocol Settings To select the method to estimate the error (Error Model)
To choose to use robust negative control statistics
208
This algorithm was developed to be used with microarrays that have many different probes classified as negative controls, such as CGH. The population outlier analysis, described on the previous pages, is performed on these negative controls, one probe sequence at a time.
Select a Robust Neg Ctrl Stats? option.
True
Once outliers are identified, they are excluded from the calculation of the robust negative control statistics (average and standard deviation).
False
See the Feature Extraction for Cyto Reference Guide for more information on how this algorithm works.
To select the method to estimate the error (Error Model)
1 Click the cell next to Choose universal error, or the most conservative (propagated or universal), and click thedownarrow.
2 Select an error method from one of these options:
Universal Error
Using this error model, a p- value is calculated that corresponds to the probability that the difference between the red and green channels is zero. The Universal Error Model is a good estimator of error in high intensity features.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select the method to estimate the error (Error Model)
Feature Extraction for CytoGenomics
It may underestimate the error of low signal features or features that have low signal- to- noise (e.g., non- uniform features).
Most Conservative
Propagated Error This method measures the error on the log ratio by propagating the pixel- level error of that feature through all the calculations on the raw signal, including raw pixel statistics, background subtraction, dye normalization and the log ratio calculation. Agilent follows standard propagation of error techniques and calculates a p- value that corresponds to the probability that the log ratio = 0.
The propagated error is a good representation of noise that predominates at low signal ranges, such as instrumentation noise and the noise due to non- uniform features.
The propagated error cannot capture other sources of noise that dominate at higher signal ranges, such as biological and chemical variability (e.g., writing and labeling, hybridization and wash processes). The propagated error method usually underestimates the noise, especially at higher signal levels.
See Parameters for Universal and Most Conservative error models" on page 209for more instructions on changing the parameters for these two error models.
Parameters for Universal and Most Conservative error models
Parameters to change With this error model and the most conservative error model, you can enter a change to the following settings and parameters:
MultErrorGreen (Red)
Enter the multiplicative error component in the channel selected
5.2 User Guide 209
4 Changing Protocol Settings To select the method to estimate the error (Error Model)
Autoestimate Additive Error
Green(Red)
210
You can select to enter your own constant for the additive error or turn on auto- estimation of the additive portion of the error model. If you select True, then Feature Extraction will auto- estimate the additive error. If you select False, then you must enter the constant.
The constant setting of the additive error approximates the error of feature signals that are near background. If the overall signal of the microarrays is increased or decreased (e.g. due to hotter or cooler labeling or variable scanner PMT settings), the additive error needs to be adjusted. The auto- estimation method of determining the additive error adjusts the scale accordingly.
CAUTION The default additive and multiplicative parameters have been
optimized using the most current Agilent microarray-based system
(e.g., microarrays, protocols, scanner and most recent Feature
Extraction algorithms). If you use non-Agilent microarrays, Agilent
microarrays with non-Agilent protocols, or Agilent microarrays with
non-Agilent scanners, you MUST re-evaluate these values to reflect
the noise in your system.
Auto-estimation of additive noise Provided that the microarray, platform and protocols remain the same, the additive error can vary from one microarray to the next. Therefore, it is useful to correctly estimate the additive noise and set the additive constant equal to this noise.
To do this the software looks at microarray statistics: the width of the negative control distribution used in the background surface fit and the spatial variability of the spatial detrend surface (RMS of the difference between each point on the surface and the mean of the surface). Because the algorithm uses these surface statistics in the calculation, this option is only usable for microarrays for which surface calculation works.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select the method to estimate the error (Error Model)
Feature Extraction for CytoGenomics
You do not have to turn on Spatial detrending for the additive error to be automatically estimated. You do have to turn on the surface fit calculation for the additive error to be estimated.
Reliability of auto-estimation of additive error Agilent has found that the auto- estimation of the additive error results in error values that are more reliable than those generated if a constant is used. If the microarray quality or processing is less than optimal, the additive error is more reliable with auto- estimation. This will make the significance calls more correct and less susceptible to errors induced by microarray and processing variability.
5.2 User Guide 211
4 Changing Protocol Settings To select the method to estimate the error (Error Model)
To choose to use or not use surrogates
212
Agilent Feature Extraction software uses surrogate signals to calculate a more accurate and reproducible log ratio for signals of low intensity.
You choose to use surrogates in this protocol step so that the signals to be used for log ratios are subjected to dye normalization to remove this kind of bias.
Agilent Feature Extraction
software uses the error estimate
option that you select for all
features, no matter if surrogates
are used for the log ratio
calculations for some features.
1 Click the cell next to Use Surrogates, and click the down arrow.
2 Select True or False.
Conditions under which surrogates are used
If you select True, the software uses surrogates under two conditions:
If either red or green MeanSignal fails the IsPosAndSignif test (i.e., IsPosAndSignif = 0)
If BGSubSignal is less than the Error
A log ratio cannot be calculated from a negative ratio. For any measurement system there is increased confidence in using a signal only if it is greater than a lower limit that is particular to that system. This ensures that the system lower limit is enforced as a lower limit on the signal.
Feature Extraction calculates the surrogate signal from the Error. For all default Agilent protocols that use Error Model significance, this is simply the Additive Error calculated during background subtraction.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change settings to test the significance of a feature
Propagation of errors from the Compute Bkgd, Bias and Error algorithm to other algorithms
Feature Extraction for CytoGenomics
The measurement errors from the background subtraction are propagated to the next steps in Feature Extraction according to standard error propagation techniques. For example, the errors for the feature and background signals are propagated with the error on the background- subtracted signal to the DyeNorm algorithm.
Estimation of error is performed in this step. See To select the method to estimate the error (Error Model)" on page 208 to see the choices show for this step. In this case, you cannot select the propagated error calculation by itself. If you select Most Conservative, Feature Extraction uses the larger of the two estimates from the Universal Error model or the propagated error.
To change settings to test the significance of a feature
Determining if a signal is significant, or just in the noise of the background, is an important step. The following two tests are performed to establish the significance of a feature: a two- sided t- test and a method to flag features that are well above background.
2-sided t-test of feature vs. background
maximum p-value
The t- statistic is calculated based on the feature and background signals and their corresponding errors.
The background value used for this test depends upon the background method used. The values are reported in the Feature Extraction text output column g(r)BGUsed. If you have chosen not to subtract the background and Spatial detrend = Off, the local background for each feature {g(r)BGMeanSignal} is used for the t- test but is not subtracted from the feature signal. If Spatial detrend = On when the background subtraction is off, the surface value for each feature is used for the t- test and is subtracted from the feature signal.
5.2 User Guide 213
4 Changing Protocol Settings To change settings to test the significance of a feature
To view the values used for the
t-test depending on the subtraction
and correction methods selected,
see the Feature Extraction for Cyto
Reference Guide.
214
Estimating background error for significance testing uses one of two methods. The method used is dependent upon the protocol parameter, Significance (for IsPosAndSignif and IsWellAboveBG).
Pixel significance
For this method, the SD of the background that is used for the t- test is the same value used for surrogates (See Use of surrogates for log ratio and p- value calculations" on page 223 of this guide). This is a population level SD for global background methods and is a pixel- level SD for both the local background and No background methods. These SD's are reported in the Feature Extraction text output columns g(r)BGSDUsed. (Not used for error model significance, which is the favored method.)
The number of degrees of freedom is also based on the error calculations and the number of pixels, which depends on the background subtraction method. The p- value is then calculated based on a two- sided t- test. If this p- value is less than the maximum p- value defined in the protocol, the feature is considered to be significantly different from the background.
Note that entering a positive pad
value for global background adjust
may increase the number of
features considered positive and
significant in the t-test, in some
cases, all features.
The features that fail the null hypothesis, that is, have a signal that is significantly different from the background and also that have background- subtracted signals > 0 will have a boolean set = 1 in the Feature Extraction text output column g(r)IsPosAndSignif. The features that are not significantly different from background or that have background- subtracted signals < 0 will have the boolean set = 0 for the same Feature Extraction text output column.
WellAboveMulti
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change settings to test the significance of a feature
Feature Extraction for CytoGenomics
Error model significance
The other method to determine feature significance is based on the error model. The protocol parameter Significance (for IsPosAndSignif and IsWellAboveBG) is set to Use Error Model for Significance for this method. The additive error is used as the estimate of background noise and is used for surrogates.
The significance test for positive and significant then becomes the probability of the feature actually having zero signal for a feature with a given background- subtracted signal with a known additive error (or noise of features with no true signal) assumed to be gaussian distributed about zero.
You choose a p- value to decide how far along the Gaussian distribution the feature needs to be before it should be considered Significant. When this choice is made, the Well Above Background is decided by a multiple of the additive error.
5.2 User Guide 215
4 Changing Protocol Settings Changing Protocol Step 6: Correct Dye Biases
Changing Protocol Step 6: Correct Dye Biases
A dye bias is the difference in the
red and green signals caused by
different efficiencies in labeling,
emission or detection.
216
Systematic differences in dye bias must be removed from the CGH data. To remove dye bias, the Feature Extraction software uses a dye normalization algorithm.
To access the settings to correct dye biases
Click the Correct Dye Biases button to change settings to remove difference in dye bias.
Figure 66 FE Protocol Editor Correct for Dye Biases selected
You can change the following types of settings for this algorithm.
Select how dye normalization gene list is determined
Select features that are included in the normalization set
Select to omit background population outliers
Select to allow positive and negative controls
Select the signal characteristics
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To choose how to select dye normalization list
Feature Extraction for CytoGenomics
Select a curve to determine the bias from that normalization set of features
The software normalizes the entire microarray from this measured bias.
If you use a self versus self experiment, in which the feature signals should be the same in both channels across the dynamic range of signal intensities, differences in dye behavior can result in the signals in one channel increasing across the signal range at a different rate than in the other channel.
This behavior can be visualized by plotting the background- subtracted signals of one channel versus the other and noting that the slope of the line fit through the data points is not equal to one. Dye normalization corrects for this by multiplying the signals by appropriate dye normalization factors.
To choose how to select dye normalization list
Three options are available for choice of the dye normalization gene list for correction of dye biases.
1 Click the cell next to Use Dye Normalization Gene List, and click the downarrow.
2 Select one of the following choices.
Automatically Determine
The DyeNormList is used, if present in the grid template. Otherwise, the list is generated internally, based on the Dye Normalization Probe Selection Method. If no DyeNormList is selected in the grid template, extraction will not fail.
True
False
5.2 User Guide 217
4 Changing Protocol Settings To choose a method to select a set of dye normalization probes
To choose a method to select a set of dye normalization probes
218
Feature Extraction supplies two methods for selecting representative features that constitute the set used for bias measurement.
1 Click the cell next to Dye Normalization Probe Selection Method, and click the downarrow.
2 Select one of the following methods.
Use Rank Consistent
Probes
This is the recommended method of selecting normalization features. The rank consistency filter selects features that fall within the central tendency of the data by observing consistent trends between the red and green channels. This method assumes that the central tendency of the data is unregulated. This method requires that the features pass the three criteria described below:
They have not been flagged as non- uniform in either channel.
They are not saturated.
The condition for saturation is more stringent in this algorithm (> 5% of pixels saturated) than in the algorithm for features as saturated (> 50% of pixels saturated).
They are not population outliers in either channel.
Rank Tolerance
To assign a red rank and a green rank, order all of the probes based on the intensity of their background- subtracted signal. A rank 1 is the lowest intensity signal feature. The highest rank is assigned to the most intense signal. A
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select to omit background population outliers
Feature Extraction for CytoGenomics
perfectly rank consistent feature would be one exactly at the 75% percentile of the distribution in both the red and green signal.
Variable Rank Tolerance
You can choose to allow variable rank tolerance. This does rank tolerance after grouping the data into signal groups. In each group the same designated percentage of most rank- consistent data are included.
Use All Probes
To select to omit background population outliers
1 Click the cell next to Omit Background Population Outliers, and click the down arrow.
2 Select True or False.
Selecting True excludes any features from the dye normalization set if the local backgrounds associated with those features have been flagged as population outliers (in either channel). The default recommendation is False.
To select to allow positive and negative controls
If you turn this function on, Feature Extraction uses positive and negative controls in the dye normalization calculation.
1 Click the cell next to Allow Positive and Negative Controls?, and click the down arrow.
2 Select True or False.
The default recommendation is False.
5.2 User Guide 219
4 Changing Protocol Settings To select signal characteristics
To select signal characteristics
220
You can select to include features with the following signal characteristics as part of the set of rank consistent features.
1 Click the cell next to Signal Characteristics, and click the down arrow.
2 Select one of the following options.
OnlyPositiveandSignificant Signals
Use only positive and significant signals for ranking in the rank consistency filter.
AllPositiveSignals
AllNegativeAndPositiveSignals
To select a normalization correction method
After you select a set of normalization features, you select a normalization method to apply to that set. The results from the calculation are applied to all features on the microarray.
1 Click the cell next to Normalization Correction Method, and click the down arrow.
2 Select from one of the following methods:
Max Number of Ranked Probes
The maximum number of ranked probes allows only a certain number of probes into the dye normalization set. If the number of rank- consistent probes exceed this number, a random set is chosen to meet the maximum number. If the number entered is - 1, this filter is not used. This option can speed up the data processing without adversely affecting accuracy for data sets with large numbers of probes, especially for 65- micron spot size and higher density arrays.
Linear
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select a normalization correction method
Linear and LOWESS
Feature Extraction for CytoGenomics
This is the default method for normalizing Agilent microarrays. Dye biases tend to be signal intensity- dependent. A dye normalization method that uses a constant scaling factor cannot correct for dye biases across the entire range of signal intensities. This method does a linear normalization across the entire range of data, then applies a non- linear normalization (see LOWESS only description below) to the linearized data set.
LOWESS only
A well- known curve fitting method called LOWESS (a locally weighted linear regression curve fit) is used to fit this curve. Given any feature and its intensity, this curve determines the potential dye bias that should be present in the log ratio measurement of the feature. Features are then corrected by multiplying the red and green channels by an adjustment that will remove the bias from the log ratio.
5.2 User Guide 221
4 Changing Protocol Settings Changing Protocol Step 7: Compute Ratios
Changing Protocol Step 7: Compute Ratios
Log ratio is the log (base 10) of the
ratio of the red channel signal to
the green channel signal.
Log ratio error is the error about
the mean of the log ratio.
P-value is a measure of confidence
that a feature is, or is not,
differentially expressed.
222
To determine if a feature is differentially expressed, three measures are important: log ratio, log ratio error and the p- value.
The log ratio error is determined by the error model and therefore used to help assess the significance of the computed log ratio.
The p- value is calculated using the log ratio and the log ratio error. Genes with lower p- values have higher confidence of being differentially expressed. For example, if the p- value is <= 0.01, there is a 99% confidence level that the gene is differentially expressed.
To access the settings to compute ratios
Click the Compute Ratios tab in the FE Protocol Editor to change the settings to calculate these measures.
See Figure 67 on page 223.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To enter the value for the log ratio limit
Feature Extraction for CytoGenomics
Figure 67 FE Protocol Editor Compute Ratios selected
You can change only one parameter for this protocol step:
Enter the value for the log ratio limit.
To enter the value for the log ratio limit
When you set a limit, the features whose log ratio exceeds that limit, either in a positive or negative direction, will not be used in any calculations. The recommended limit for this value is 4.
1 Click the down arrow in cell next to Peg Log Ratio Value.
2 Enter a positive or negative value up to 4.
Use of surrogates for log ratio and p-value calculations
Surrogates are used in this step to calculate certain log ratios, p- values and log ratio errors.
The table below shows the conditions for use of surrogates to calculate log ratios, log ratio errors and p- values.
5.2 User Guide 223
4 Changing Protocol Settings Use of surrogates for log ratio and p-value calculations
*For log ratios equal to or between -4 (ratio = .0001) and +4 (ratio = 10000). If (r/G) > 1 or (R/g) < 1, log ratio = 0 and p-value = 1.
Table 8 Use of surrogates for log ratio and p-value calculations
If red channel signal is And green channel signal is Then ratio is
And Feature Extraction does this*.
Positive and significant vs.
background and greater than
red background SD (R)
Positive and significant vs.
background and greater than
green background SD (G)
R/G Uses the dye-normalized signals for both
channels for the calculations.
Not positive and significant vs.
background or less than red
background SD (r)
Positive and significant vs.
background and greater than
green background SD (G)
r/G Uses the dye-normalized green signal and
the dye-normalized red background SD signal
(surrogate) for the calculations.
Not positive and significant vs.
background or less than red
background SD (r)
Not positive and significant vs.
background or less than green
background SD (g)
r/g Uses surrogates for both channels.
Sets the log ratio = 0.
Sets the p-value = 1.
Positive and significant vs.
background and greater than
red background SD (R)
Not positive and significant vs.
background or less than green
background SD (g)
R/g Uses the dye-normalized red signal and the
dye-normalized green background SD signal
(surrogate) for the calculations.
Table 9 Summary Use of surrogates for calculations
Case 1: R/G
Both channels use DyeNorm Signals.
P-value and log ratio are calculated as usual.
For signals not using surrogates,
g(r)DyeNormSignal = g(r)ProcessedSignal,
which is then used to calculate log ratio.
Case 2: r/G
r = rSurrogateUsed
G = gDyeNormSignal
P-value and log ratio are calculated as usual.
If r/G > 1, then Feature Extraction automatically sets
LogRatio = 0 and PvalueLogRatio = 1
Case 3: R/g
R = DyeNormSignal
g = gSurrogateUsed
P-value and log ratio are calculated as usual.
If R/g < 1, then Feature Extraction automatically sets
LogRatio = 0 and pValueLogRatio = 1
Case 4: r/g
Both channels use surrogates.
Feature Extraction automatically sets
LogRatio = 0 and pValueLogRatio = 1
For signals using surrogates,
g(r)ProcessedSignal =
g(r)SurrogateUsed x g(r)DyeNormFactors.
224 Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 Use of surrogates for log ratio and p-value calculations
Feature Extraction for CytoGenomics
Effect of using surrogates on log ratios
Below are plots that show the difference between using surrogates and not using surrogates for a set of self vs. self microarray data.
Figure 68 Plot of LogRatio vs. Avg_grProcessedSignalNo surrogates
Green stars are data points affected by use of surrogates in Figure 69.
5.2 User Guide 225
226
4 Changing Protocol Settings Use of surrogates for log ratio and p-value calculations
Figure 69 Plot of LogRatio vs. Avg_grProcessedSignalWith surrogates
Green stars are data points affected by use of surrogates. See Figure 68 for their positions without surrogates.
Effect of using surrogates on log ratio errors
If Use Surrogates is True, the software calculates log ratio errors using the dye- normalized and/or surrogate signals, according to the error model selected for the calculation.
If Use Surrogates is False, the software calculates log ratio errors using the dye- normalized signals, according to the error model selected for the calculations. If either or both channels have negative dye- normalized signals, the p- value is set to 1 and the log ratio error is set to 1000. If red channel < 0, then Feature Extraction sets the log ratio = - 4. If green channel < 0, then Feature Extraction sets the log ratio = +4. If both red and green channels < 0, then Feature Extraction sets the log ratio = 0.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 Changing Protocol Step 8: Calculate Metrics
Changing Protocol Step 8: Calculate Metrics
Feature Extraction for CytoGenomics
In this step you can change the settings for the metrics that appear in the QC report.
To access the settings to calculate metrics
Click the Calculate Metrics tab in the FE Protocol Editor.
Figure 70 FE Protocol Editor Calculate Metrics selected
You can change the following parameters in this step:
Select True for Spikein Target Used if Agilent spike- in targets were used during lab processing of the microarray. The appropriate metrics are generated in the QC report.
Enter the minimum population for replicate statistics.
Select the grid test format.
Enter the p- value for differential expression.
Enter the percentile value.
5.2 User Guide 227
4 Changing Protocol Settings To select to use spikein targets
To select to use spikein targets
228
1 Click the cell next to Spikein Target Used?, and click the down arrow.
2 Select True or False.
If you select True, Feature Extraction will do all the calculations necessary to report the statistics for both the non- control features and the spikein controls.
To change the minimum population for replicate statistics
The QC report includes statistics based on replicate probes (%CV, log ratio uniformity). Feature Extraction must know the minimum population of replicates in order to calculate these statistics.
1 Click the cell next to Minimum Population for Replicate Statistics.
2 Enter a number of 3 or greater.
To select the grid test format
1 Click the cell next to Grid Test Format.
2 Select the grid format of the images to be tested, or select Automatically Determine to let the software decide for you.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To change the p-value for differential expression
To change the p-value for differential expression
Feature Extraction for CytoGenomics
The QC report contains a plot of the log ratio vs. the magnitude for each feature to show which features are up- regulated, which are down- regulated and which show no differential expression. This assessment is relative to the p- value chosen for this setting.
1 Click the cell next to PValue for Differential Expression.
2 Enter a p- value against which you intend to measure differential expression on the microarray.
To change the percentile value
This value is an indicator of the distribution of signals of the non- control features and is useful in downstream normalization of 1- color data.
1 Click the cell next to Percentile Value.
2 Enter a value to set the distribution of signals of the non- control features.
5.2 User Guide 229
4 Changing Protocol Settings Changing Protocol Step 9: Generate Results
Changing Protocol Step 9: Generate Results
To access the settings to generate results
230
Click the Generate Results tab in FE Protocol Editor to change the values for output.
Figure 71 FE Protocol Editor Generate Results selected
You can change the following settings for this protocol step:
Select the QC Report type.
Select to generate a single file for the text output.
Change the compression factor for the JPEG output.
To select the type of QC Report
1 Click the cell next to Type of QC Report, and click the down arrow.
2 From the list, select the QC report type to be generated.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Protocol Settings 4 To select to generate a single file for the text output
Gene Expression
Feature Extraction for CytoGenomics
Generate a Gene Expression QC report.
Streamlined CGH
Generate a Streamlined CGH QC report. For more information, see the Feature Extraction for Cyto Reference Guide.
CGH_ChIP
miRNA
Test Chip
To select to generate a single file for the text output
1 Click the cell next to Generate Single Text File?, and click the down arrow.
2 Select True or False.
If you select true, the text output appears in a single file. If you select False, it appears in three separate files, one for FEPARAMS, one for STATS and one for FEATURES (results).
To change the compression factor for the JPEG output
The value for this factor determines the degree to which the image is scaled down in both directions before being converted to JPEG.
1 Click the cell next to JPEG Down Sample Factor, and click thedownarrow.
2 Enter a number.
5.2 User Guide 231
232
4 Changing Protocol Settings To change the compression factor for the JPEG output
Feature Extraction for CytoGenomics 5.2 User Guide
Agilent CytoGenomics 5.2 Agilent Feature Extraction for CytoGenomics User Guide
5 Changing Image Displays
Displaying and saving image files 234
Loading Feature Extraction visual results 240
Changing image display settings 247
Changing Feature Extraction window displays 269
This chapter presents instructions on changing image displays and working with image files to view the images before and after extraction.
233Agilent Technologies
5 Changing Image Displays Displaying and saving image files
Displaying and saving image files
To open an image file
234
1 Click on the toolbar Open image file button, or click File > Open > Image, to display the Open Dialog Box.
2 In the Open Dialog Box, double- click on a .tif file from the scanner, or select a file and click Open.
You can also open an image file by
dragging the file to the Feature
Extraction desktop icon.
An image of the scanned microarray contained in the file appears in the Workspace.
Figure 72 Uncropped TIFF image for Agilent microarray
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To crop an image
Feature Extraction for CytoGenomics
You can also open an image file double- clicking the image file in a project extraction set. For information on how to do this, see Chapter 2, Extracting Microarrays Automatically.
To crop an image
Crop an image using the cursor
You do not have to crop an Agilent image before you load it into a project and extract. You may want to crop images to view a subset of features or to use for illustration purposes.
When you first open an image, the
Cropping Mode button is turned
ON because this is the default
setting. (See To change default
image viewing settings" on
page 263.)
1 If not already on, click the Cropping Mode On/Off button.
2 Position the crop cursor at the top left corner of the image that you want to crop, and hold the left mouse button down.
3 Drag the crop cursor to the opposite corner.
4 Release the left mouse button to display the cropped image.
Figure 73 Image top menu and toolbar in Feature Extraction Tool
Cropping mode ON
The Status Bar is located at bottom
of the Feature Extraction window
and contains the pixel information
about the viewed image. If the
viewed image is the preview
image, the pixels are from the
preview. If the viewed image is the
high-resolution image, the pixels
are the high-resolution pixels.
If you are viewing high- resolution images, you must crop them to one- tenth or less (i.e. at least 200%) of their original size in order to see the features at high resolution. Any larger cropped image is a preview image.
The color of the crop cursor helps you recognize the proper size for the high fidelity image. If the cursor is black, the cropped image is the high fidelity image. If the cursor is red, the cropped image is still a preview image.
5.2 User Guide 235
236
5 Changing Image Displays To crop an image
You can cancel the cropping operation by using the (Esc) key, or click the Cropping Mode On/Off button to turn cropping off.
Crop an image using corner locations (manual crop)
This option lets you crop an image with greater precision than you can attain with the cursor option.
NOTE For manual cropping to work, the image after cropping must be a minimum
of 30 x 30.
1 Click Edit > Manual Crop.
Figure 74 Manual Crop dialog box
2 Enter the locations of the corners (diagonal to one another) that you intend to have as the corners of the cropped image.
3 Click OK.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To crop an image
To save a cropped image to a new file
Feature Extraction for CytoGenomics
If the image file is from an Agilent microarray with a barcode:
1 Click File > Save Modified Image to save the cropped image to a new file.
Figure 75 Save Modified Image dialog box for Agilent microarray
2 To close the file after saving, clear the Open file after saving check box.
3 To specify which pack in the multiplex (formerly known as multipack) microarray was cropped, select a Multiplex_Row_Column number.
This lets you save the cropped single pack with its multiplex coordinates.
If no multiplex was cropped, clear the Cropped Multiplex check box.
4 To change the New Array Identifier, see To add or change the New Array Identifier" on page 239.
5.2 User Guide 237
238
5 Changing Image Displays To crop an image
For Agilent microarrays with a barcode, the New Array Identifier is automatically generated from the barcode and displayed, along with a suggested file name.
The New Array Identifier can be changed. In fact, if for any reason the barcode does not generate a New Array Identifier, you must enter one if you intend to send the MAGE- ML results file to Resolver.
5 To change the path for the cropped image:
Click the ellipsis (...)button.
Select the new path.
Click OK
6 Click Save Modified Image.
If the image file is from a non-Agilent microarray:
Click File > Save Modified Image to save the cropped image to a new file.
For microarrays without an identifier, the New Array Identifier field is blank. You can add an identifier. See To add or change the New Array Identifier" on page 239.
Figure 76 Save Modified Image dialog box for non-Agilent microarray
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To crop an image
To add or change the New Array Identifier
Feature Extraction for CytoGenomics
When you attempt to save a modified image, the Save Modified Image dialog box appears with a field for New Array Identifier. Agilent barcodes automatically generate this number. For non- Agilent images, this field is blank.
If the New Array Identifier is not generated, for whatever reason, the software will not transfer MAGE- ML or GEML results files to a third- party program such as Resolver.
You can change or add this identifier in the Scan Image Properties dialog box.
1 Click View > Image File Info to bring up the Scan Image Properties dialog box.
2 Click the cell next to the Identifier field, and enter the new number.
3 Click Apply, and click Close.
A message appears that says you must click File > Save As before the change is accepted.
4 Click OK, and click File > Save As.
5 Enter a new name or select the old name of the .tif file, and click Save.
If you were trying to save a modified image, try again. The New Array Identifier you entered appears in the field.
5.2 User Guide 239
5 Changing Image Displays Loading Feature Extraction visual results
Loading Feature Extraction visual results
240
You can select to store visual results in the same local folder as the other types of result files or to have it be sent via FTP to a remote directory. In either case the visual results file is saved with the same name as the results file name with an extension of .shp.
By default, the software shows the results for outlier features only when you load the visual result file or shape file (.shp) for the image. To view all results, you must clear the View Outliers Only check box. It is recommended, the you first crop out a region of the image before clearing the outlier check box. For instructions, see To change the magnification (zooming)" on page 255 and To change the extraction results display" on page 258.
1 Open the image for which you want to see the visual result.
Figure 77 Image of Agilent 1-color, 4x44K microarray
2 Click Feature Extraction > Load Visual Result to load previously saved visual results for the currently displayed image file.
The software opens the folder containing the image file whose visual results you want to view. If you chose to save the output files for the project to a results folder separate from the image file folder, you must move to that folder.
3 Select the shape file, and click Open.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To view visual results for low-resolution images:
Feature Extraction for CytoGenomics
Or, you can also open the visual result file that is associated with the extraction set from the Project Explorer.
1 From Project Explorer, right- click the image.
2 Click Load Visual Results.
This opens the image and loads the shape file associated with that extraction set.
To view visual results for low-resolution images:
1 After .shp file is loaded to the image, zoom in until you see enough of the spots to help you evaluate the results.
2 Pass cursor to feature of interest and hold for a few seconds.
A description of the feature appears.
To find a specific feature, use Ctrl-F.
Enter the Feature number of the
specific feature you want to find.
Figure 78 Visual results for low-resolution microarray image
5.2 User Guide 241
5 Changing Image Displays To view visual results for low-resolution images:
To view visual results for high-resolution images (2- by 3-micron or 20-bit):
242
After you first load the image, you, in fact, are viewing a preview image. You can only view the original high- fidelity image if you crop the size of the image to one- tenth or less of the original size.
Figure 79 Preview display of high-resolution image with outliers in blue
1 After .shp file is loaded to the image, zoom in until you see enough of the spots to help you identify the region of interest.
2 Click the left mouse button at the point where you want to start forming a rectangle for cropping.
3 With the crop cursor, draw a rectangle equal to or less than one- tenth the size of the image.
Note that the crop cursor is black if the size is within one- tenth (200% to 1000%) of the original size, but it turns red if you are drawing outside the high fidelity range.
4 Then release the mouse button.
Note the status bar at the bottom of the window. It should now read High Fidelity.
High fidelity mode is when you zoom in the image by at least 200%.
5 Pass cursor to feature of interest and hold for a few seconds.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To view visual results for low-resolution images:
Feature Extraction for CytoGenomics
A description of the feature appears.
Figure 80 Visual results of high-resolution microarray image in
High Fidelity mode, zoomed to 500%
View pixels used
This view option for low- resolution images and for high- resolution images that are in High Fidelity mode (i.e. when the image is zoomed in to one- tenth of its original size).
The View Pixels Used option displays the pixels used to quantify the feature signal within the cookie area. Pixels that are determined to be outliers are shown in black.
1 If the image is a high resolution image, complete steps 1- 4 from To view visual results for high- resolution images (2- by 3- micron or 20- bit):" on page 242.
2 Under View > Extraction Results, select View Pixels Used.
5.2 User Guide 243
244
5 Changing Image Displays To view visual results for low-resolution images:
Figure 81 View pixels used for high-resolution microarray image in
High Fidelity mode, zoomed to 500%
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To view visual results for low-resolution images:
To save an image (original or cropped) for import to another program
Feature Extraction for CytoGenomics
1 Click File > Save As to produce high resolution images.
The Save As dialog box lets you save .tif files or .jpg files of the current image or cropped image.
2 Click the down arrow to the right of the Save as type: list.
3 Select either Tif Files, BMP files Png Files, or Jpeg Files.
4 Enter the name of the image.
5 Click Save.
Figure 82 Save As dialog box
5.2 User Guide 245
5 Changing Image Displays To view visual results for low-resolution images:
To display file information
246
You can use the File Info dialog box to view valuable information, such as scanner warning messages, scanner version, PMT voltage, dark offset information, the scan resolution and glass thickness
Click View > Image File Info to display a tabbed dialog box that displays all available information about an image file.
Figure 83 Scan Image Properties dialog box
The only field that you can change is the Identifier field. See To add or change the New Array Identifier" on page 239.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 Changing image display settings
Changing image display settings
Changing the image display does
not affect the Feature Extraction
results in any way.
Feature Extraction for CytoGenomics
When you open an image you can manipulate displayed images from the original scan file, the cropped scan file or the Feature Extraction results in the following ways:
Change image orientation (landscape/portrait)
Change the number of channels displayed
Change the display color
Change between log and linear color scale
Change the intensity scale (color display range)
Change the magnification (zooming)
Change the fit of the window to the image
Change the extraction results display
This section also shows you how to change settings for histograms and line graphs, the default image analysis parameters and printing.
To change image orientation (landscape/portrait)
Due to the size of 2um or 3um scan
images, the flip and rotate
operation can take several
minutes.
When you first load a microarray image into the Feature Extraction Tool, the image appears in the orientation produced by the scan, either landscape or portrait. If the gene list that you want to use to position a grid and find spots has a different orientation than the image, you must change the image orientation to match that of the gene list.
Flip and rotate of 2um, 3um, and
20-bit scans is supported in v10.7
and above.
1 Click Tools > Flip Upper Left to Lower Right (Landscape/Portrait) from the top menu.
If the original orientation was landscape, the software takes the upper left corner and moves it to the lower right corner to create a portrait orientation. The same is true if you go from a portrait to landscape orientation.
2 Click File > Save modified image.
If you do not save the image in its new orientation, you could run into problems with Feature Extraction later.
5.2 User Guide 247
5 Changing Image Displays To change the channels that display
To change the channels that display
248
The default setting for displaying the channels is to display one window with the two color channels combined.
To change the channels for a single window:
1 On the image viewing toolbar click the down arrow for the Channel Display menu.
The default selection is All Channels.
2 Select Red Channel or Green Channel.
The window display changes to the channel selected.
You can also change the number and color of the channels displayed when you set default image settings.
To display separate channel images:
Displaying the images of separate color channels side by side can be useful for comparing background anomalies on the microarray.
1 Click Window > New Filename View Window.
Filename represents the path and name of the image that you loaded. The Select Image dialog box appears.
2 Click Red or Green.
3 Click OK.
If you repeat steps 1- 3, all three windowsthe red, the green and the combined colordisplay at the same time.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change the channels that display
To change the display color
Color Mode choices are False
Color, Gray Scale, Reverse Gray
Scale and False Color w/White BG.
Feature Extraction for CytoGenomics
From the image viewing top menu, click Color > color mode.
Figure 84 Color menu
The color type that you select depends on the channel image display. For combined channels, there are two choices of color, and for single channels there are three choices.
Combined channel display
The default setting for the color on the TIFF image is called False Color. You also have the option of selecting False Color with a white background.
Click Color > False Color w/ White BG.
To return to False Color, click Color > False Color.
False Color
False Color w/ White BG
This selection maps intensities the same as the False Color palette, except that a pixel with zero values in both red and green is turned white instead of black. This feature is useful when printing because it saves ink. Generally, the minimum data range must be manually adjusted to produce the best possible image.
5.2 User Guide 249
5 Changing Image Displays To change the channels that display
250
Single channel display
The default setting for the color on the single- channel display is also False Color.
Click either Color > Gray Scale or Color > Reverse Gray Scale.
Gray Scale
Reverse Gray
To return to False Color, click Color > False Color.
Examples of images with different color selections
The following images are intentionally not aligned.
Figure 85 False Color for 2-color image
Figure 86 False Color with white BG for 2-color image
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change the channels that display
Feature Extraction for CytoGenomics
Figure 87 False Color for 1-color image
Figure 88 Gray Scale
Figure 89 Reverse Gray Scale
5.2 User Guide 251
5 Changing Image Displays To change the intensity scale (color display range CDR)
To change between log and linear color scale
252
Click Color > Use Log Color Scale to toggle the color scale from linear to log10 (intensity).
To return to the linear scale, click Color > Use Log Color Scale again to remove the check mark.
With the log scale you can see low intensity portions of the microarray at the expense of detail at high intensities.
You can also use the Log/Linear button on the toolbar to toggle the color scale.
To change the intensity scale (color display range CDR)
Changing the color display range affects how the image intensity is scaled. Modifying the color display range can make it easier to visualize your microarray image but does not affect the raw intensity.
You can change the color display range in one of two ways:
Using the color scaling toolbar.
Using the Set Color Display Range dialog box.
Use the color scaling toolbar
The easiest way to determine the best range is to use the Auto Scalefunction on the toolbar.
Figure 90 Color scaling segment of the image viewing toolbar
CDR Low Mode Icon CDR High Mode Icon
Feature Extraction for
Changing Image Displays 5 To change the intensity scale (color display range CDR)
Feature Extraction for CytoGenomics
1 Click the down arrow next to the color scaling menu, and click Auto Color Scaling if necessary.
2 Click the CDR Low Mode icon, and move the lower bar cursor to the minimum percentage for the display range.
3 Click the CDR High Mode icon, and move the higher bar cursor to the maximum percentage for the display range.
Notice the change in color of the image display as you increase or decrease the blue bar size.
4 To fine tune the color display ranges manually, click either the Red Channel or the Green Channel from the color scaling menu.
5 Click the CDR Low Mode icon, and move the lower bar cursor to the minimum pixel number for the display range.
6 Click the CDR High Mode icon, and move the higher bar cursor to the maximum pixel number for the display range.
5.2 User Guide 253
254
5 Changing Image Displays To change the intensity scale (color display range CDR)
Use the Set Image Color Display Ranges dialog box
Alternatively, you can set the color display range through the Set Image Color Display Ranges dialog box.
1 Click Color > Set Color Display Range, or click Set Color Range on the toolbar. .
Figure 91 Set Image Color Display Ranges dialog box
2 Mark the Auto Scale Image check box to set a low and a high percentage cutoff value.
Setting 0 for a low value and 100 for a high value will compress the entire range of possible values into the 8- bit displayable range. If this produces a black image, try 1 and 99 (the default settings). Generally, this gives you an acceptable data range, even for very poor images.
3 To fine tune the color display ranges manually, clear the Auto Scale Image check box.
4 Change the Minimum and Maximum Red and Green values.
The data range determines how the 16- bit image data is mapped to the 8- bit data that is displayed on a computer screen. Generally, scanned images do not cover the entire range of possible values: 0 to 65535. Therefore, if you try to display the entire range, all you can see is black.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change the magnification (zooming)
To change the magnification (zooming)
To set the default % zoom settings
for the loaded image and the grid
mode images, see To change
default image viewing settings" on
page 263 of this guide.
Feature Extraction for CytoGenomics
Magnification can only be set to one of the % zoom levels shown in Figure 92. Numbers greater than 100% map one scan pixel to multiple pixels on the screen. For example, 600% maps one scan pixel to a 6 x 6 square of identical pixels. A zoom value of 100% maps one scan pixel to one pixel. Values less than 100% display the mean value for several scan pixels to one pixel. For example, 50% averages four scan pixels, a 2 x 2 matrix, and displays the average as one pixel.
You can control zooming in one of three ways:
Dragging the cursor to make a rectangle
Using buttons on the tool bar and on the mouse
Using the View menu
Drag the cursor to make a rectangle
You can only zoom the image using this method if cropping is turned off.
1 Clear the Cropping Mode On/Off button to turn cropping off.
2 Position the cursor at the top left corner of the image section that you want to zoom, and hold the left mouse button down.
3 Drag the cursor to the opposite corner of the section you want to zoom.
4 Release the left mouse button to display the selected area.
Cropping mode OFF
5.2 User Guid
256
5 Changing Image Displays To change the magnification (zooming)
Use the Tool Bar and Mouse Buttons
Three buttons on the tool bar and the right and left mouse buttons control zooming. You can use these buttons with Crop Mode On or Off.
Use the Zoom Menu
You can use the Zoom menu with Crop Mode On or Off.
Click View > Zoom to jump to any zoom level directly.
For low- resolution images, if the image is uncropped, the zoom levels range from 10% to 1000%.
For high- resolution images, if the viewed image is uncropped, the zoom levels range from 10% to 100%. To activate zoom levels from 200% to 1000% (High Fidelity mode), you must crop the image.
Note that the crop cursor is black if the size is within one- tenth (200% to 1000%) of the original image size, but it turns red if you are drawing outside the High Fidelity range.
Figure 92 Zoom menu for low-resolution image
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change the window size to fit the image
To change the window size to fit the image
Feature Extraction for CytoGenomics
If you have to scroll to see the entire image, use this option to increase the size of the window to fit the image. If the window is too big for the image and contains white space around the perimeter of the image, use this option to decrease the size of the window to fit the image.
Click View > Entire Image to resize the window.
5.2 User Guide 257
5 Changing Image Displays To change the window size to fit the image
To change the extraction results display
258
Use this option to control the portions of the feature extraction results that are displayed.
Click View > Extraction Results.
Figure 93 Extraction Results Default views selected
View Results
Shows all visual results when this option is selected and View Outliers Only is not selected.
Shows outlier results only when both this option and View Outlier Only are selected.
Hide all visual results when this option is not selected, even if View Outliers Only is selected.
View Outliers Only
Hide Outer Local BG Ring
Shows or hides the local background ring. See To select a spot statistics method to define features" on page 187 of this guide for a definition of the local background.
Use Simple Colors
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change and save histograms and line graphs
View Pixels Used
Feature Extraction for CytoGenomics
Shows or hide pixels within the cookie that are used to quantify the feature mean or median intensity.
For more details, see View pixels used" on page 243.
To change and save histograms and line graphs
For high- resolution images, histograms and line graphs use the original data, not the data from a low- resolution preview image.
Set up a histogram graph
Histograms can be very useful to initially visualize your results. They can show you if you have an anomalous distribution of intensities across the entire slide or across rectangular or elliptical subregions of the image.
Entire viewing area
1 Click View > Entire Histogram from the main menu bar.
If the selected file has more than one color, a Channel Selection dialog box appears.
2 Select the desired channel, or select both.
Rectangular subregion
1 Make sure that cropping is OFF.
2 Click the right mouse button to position your cursor in one corner of the region to capture.
3 Hold down the right button and drag the pointer to the opposite corner, then release.
If the selected file has more than one color, a Channel Selection dialog box appears.
4 Select the desired channel, or select both.
5.2 User Guide 259
260
5 Changing Image Displays To change and save histograms and line graphs
Elliptical subregion
1 Click Tools > Preferences > Graph Viewing.
2 Click the cell next to Select Elliptical Area for Histogram, and click the Up arrow.
3 Select True, and click OK.
False is the default setting and results in a rectangular subregion.
Figure 94 Histogram Preference Settings dialog box
4 Make sure that cropping is OFF.
5 Click .
Or, click View > Select Elliptical Area.
6 Click the right mouse button to position your cursor in one corner of the region to capture.
7 Hold down the right button and drag the pointer to the opposite corner, then release.
If the selected file has more than one color, a Channel Selection dialog box appears.
8 Select the desired channel, or select both.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change and save histograms and line graphs
Feature Extraction for CytoGenomics
Change the bin size for histograms
1 To adjust the bin size after creating the initial graph, click Histogram > Change Bins.
2 Change the bin size or the maximum number of bins.
3 Click OK.
The maximum bin size is 65535; the minimum is 1.
The maximum number of bins is 32768; the minimum is 2.
Change the default bin size for histograms
1 Click Tools > Preferences.
2 Click the Graph Viewing folder.
3 Change the bin size or the maximum number of bins.
4 Click OK.
5.2 User Guide 261
262
5 Changing Image Displays To change and save histograms and line graphs
Set up a line graph
Line graphs show a vertical or horizontal slice through the image. They can be useful for spotting extreme differences between the red and green signal intensities across the microarray. Line graphs are also useful for displaying average intensity over all rows and columns, minimizing the effect of noise.
To create a line graph more quickly,
double-click a selected row within
the image with the left mouse
button, or double-click a selected
column with the right mouse
button.
1 Click View > Select Row or Column, or click or .
The Select Row/Col dialog box appears.
Figure 95 Select Row/Col dialog box
2 Click Row Number for showing signals in a row, or click Column Number for showing signals in a column.
3 Enter the number of the row or column.
4 Mark the All Rows or Columns check box to average the rows or columns in the view depending on the Row Number or Column Number selection.
5 Click OK.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change default image viewing settings
Feature Extraction for CytoGenomics
Figure 96 Line graph example
Use the Graph Toolbar
You save graphs produced from the image files to third party programs, such as word processors and spreadsheets, with the buttons in the Graph Toolbar. You can also print and modify the graph display with this toolbar.
Click the selected button in the Graph Toolbar at the top of the histogram or line graph.
To change default image viewing settings
You can also change the default settings that appear when you open a new file. To activate the settings you must exit the image file and either reopen the same file or open a new one.
5.2 User Guide 263
5 Changing Image Displays To change default image viewing settings
See To change the display
color" on page 249, To change
between log and linear color
scale" on page 252, and To
change the intensity scale (color
display range CDR)" on page 252
in this chapter for details on the
selections in the first two sections
of the Image Viewing Preferences
window.
264
1 Click Tools > Preferences.
2 Open the Image Viewing folder, if necessary.
3 Change the default selections if you need to.
4 Click OK.
5 Close the image.
6 Reopen a file.
The new default settings now take effect.
Figure 97 Image View Default Options dialog box
Color Palette
Use log color scale
If set to True, the software uses the log10 intensity scale for the color. If False, it uses a linear scale.
AutoScale
Start with crop mode
Select True if you want to be able to crop the image with mouse movement right after you load the image. Select False if you want the mouse to activate the zoom function.
Ratio of window size to feature cross for found
feature
When you attempt to find a feature in the visual result image, you can change the ratio of the resulting window size for the image to the size of the feature cross for this found feature.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To change the print settings
Feature Extraction for CytoGenomics
Change other default settings in the Preferences dialog box
See the tasks in the relevant chapters for changing the default settings for the Grid Mode (Chapter 2), for Graphs (histogram bin settings in this chapter) and for Projects (Chapter 1).
To change the print settings
The Feature Extraction application supports printing to both color and black and white printers.
Change page settings
Click File > Page Setup to set the following printer paper parameters:
Paper size
Paper source
Paper orientation
Margins
Printer
Change print settings
1 Click File > Print Setup or File > Print to bring up the standard Windows Print Setup dialog box.
Within this dialog you can change the following parameters:
Paper size
Paper source
Paper orientation
Margins
Printer
2 Click OK on the Print Setup dialog box to save changes to the printer setup without printing, or
5.2 User Guide 265
266
5 Changing Image Displays To change the print settings
Click Print to save changes to the printer setup and to print.
Preview a file before printing
Click File > Print Preview.
Printing single color images
If you are printing a single color image that is displayed using the false color palette to a black and white printer, the Image Analysis Tool of Feature Extraction automatically converts the image to gray scale. This is necessary because the gray scale intensities that a black and white printer automatically generates for a false color image are incorrect.
Examples
Figure 98 False Color image
Figure 99 Incorrect image generated by blank and white printer
Figure 100 Correct gray scale generated by the Image Analysis Tool
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 Hot Key actions for image displays
Feature Extraction for CytoGenomics
Printing 2-color images
Printing 2- color images on a black and white printer does not require the Image Analysis Tool to convert the image to gray scale, but you cannot distinguish between the colors. See the following example.
These images are intentionally not
aligned.
Figure 101 2-color image
Figure 102 2-color image printed on a black and white printer
Images always print at maximum resolution. The zoom level does not affect printing. This resolution setting makes printing the image slow but also generates the highest quality image possible.
Hot Key actions for image displays
Table 10 Hot key actions for image displays
If you want to do this: Press this hot key:
Turn Crop Mode On/Off Alt-C
Call up the manual crop dialog box Ctrl-M
Call up the File Info dialog box Ctrl-I
Change image orientation
(landscape/portrait)
Shift-U
Zoom in Ctrl-double-click left mouse
5.2 User Guide 267
268
5 Changing Image Displays Hot Key actions for image displays
Find feature when viewing extraction
results
Ctrl-F
Open new window
Does not work for high resolution
scans (2um, 3um, or 20-bit)
Ctrl-N
Open file Ctrl-O
Print TIFF image Ctrl-P
Save the image as a different file Ctrl-S
Table 10 Hot key actions for image displays
If you want to do this: Press this hot key:
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 Changing Feature Extraction window displays
Changing Feature Extraction window displays
Feature Extraction for CytoGenomics
When you start the Feature Extraction software, you see the default window layout that Agilent configured before shipment. This window layout always appears unless you move windows and panes and change the default settings in the Preferences dialog box.
Figure 103 FE Standard Project Configuration window default
This section shows you how to change from one layout to a new layout and to save the new layout so that it appears when the software starts.
5.2 User Guide 269
5 Changing Image Displays Changing Feature Extraction window displays
To move windows and panes
270
Windows can be separated from window frames and blended into frames (maximized).
Click the Restore Window icon for the Project Work Window. See Figure 103.
The Project Window becomes separated from the main window frame, exposing the Workspace.
Panes can be unattached from one edge of a window frame and docked to another edge of the frame.
1 Click on the title of the Grid Template Browser Pane and drag it from the right edge of the window frame.
2 Move the Browser across the Workspace until the cursor hits the left edge of the frame, then release the mouse button.
The Grid Template Browser is now docked to the left edge of the frame. You can dock a pane to any edge of the frame in this way.
3 Repeat step 1 for the FE Protocol Browser Pane.
4 Move the Browser across the Workspace and place the cursor on the title of the Grid Template Browser.
Now both panes are overlapping one another with tabs at the bottom of the panes. To separate the two panes once more, click on a tab and move it to a new position.
If you had moved the FE Protocol Browser Pane to the left edge of the frame, the two Browsers would be next to each other.
Click on the title of the Project Explorer Pane and drag it across the Project window until the cursor hits the right edge of the window frame, then release the mouse button.Two other actions can be taken with panes:
Close panes. Click on the X in the upper right corner.
Autohide panes when docked. Click on the pin icon in the upper right corner of the pane.
The pane now appears as a bar button on the edge to which it was docked.
Feature Extraction for CytoGenomics 5.2 User Guide
Changing Image Displays 5 To select the layout to use when software starts
Feature Extraction for CytoGenomics
When you click on the resulting bar button, the pane floats from the edge so that you can view its contents. Note that the pin position is now horizontal instead of vertical. If you click on the pin now, the pin becomes vertical and the pane redocks itself to the edge of the frame.
To select the layout to use when software starts
You set the default layout for the Feature Extraction display in the Preferences dialog box.
1 Click Tools > Preferences from the top menu.
2 Change the following settings to produce the layout you want upon software startup.
If you want to return the windows
and panes to their original
positions at any time, set Frame
Windows Using last saved
Position and Docking Pane Using
last saved Layout to FALSE, close
Feature Extraction and reopen the
software.
Figure 104 Preference Settings dialog box
Frame Windows Using last saved Position
If True, the window positions you have changed in the last session appear upon startup. If False, the factory default window position appears.
5.2 User Guide 271
5 Changing Image Displays To select the layout to use when software starts
Docking Pane Using last saved Layout
272
If True, the docking positions from the last session appear upon startup. If False, the factory default docking positions appear.
Initially Loading Project
If New Project, FE Project1 always appears upon startup. If Last Project, the project on the screen before exiting appears at startup. If None, the Workspace is blank upon startup.
Feature Extraction for CytoGenomics 5.2 User Guide
A
additive error, 208 algorithm
purpose, 29
B
background correction adjust background globally, 206 background subtraction method, 201 multiplicative detrending, 201 no background subtraction, 201 spatial detrending, 204 surface fit calculation, 204
background subtraction methods global, 201 local, 201
barcode id, add to tiff header, 239 barcode, need for data transfer, 99
C
calculate spot size and centroids, 144 channels
changing display, 248 colors
changing log/linear scale, 247, 252 changing the display, 248 examples of selections, 249
Cookie Cutter method, 187 correct dye biases
normalization correction method, 220 positive and negative controls, 219 select probes, 218 select signal characteristics to determine rank consistency, 220
D
default settings grid mode, 157
default settings, changing for Image Viewing, 263 definitions
dye bias, 216 feature, 183
Feature Extraction for CytoGenomics 5.2 User Guide 273
log ratio, 222 log ratio error, 222 negative controls, 203 p-value, 222 spot, 183
differential expression p-value, 229
display settings changing colors, 263 changing defaults, 263
dye bias definition, 216 removing differences, 216
dye normalization algorithms, 220 methods of curve fitting, 220 selecting set of features, 218 what algorithm does, 220
DyeNorm gene list change default, 80 create, 80 create external, 84 edit, 84 Editor, 81 export, 84 external file, 50 import, 81 save, 84
E
error models combination, 222 propagated error, 222 Universal, 222
error propagation correct bkgd and signal biases, 213 find and measure spot, 190
extraction results display, changing, 258 extraction results, viewing, 257 extraction sets
access grid mode, 115 access grid mode from, 77 create new project if run fails, 114 create project if run fails, 93 definition, 67 view number in project, 45
274 Feature Extraction for CytoGenomics 5.2 User Guide
F
Feature Extraction 1-color run, 93 monitor runs, 63 number set to run simultaneously, 63 start, 93 stop, 93 terminate, 44 turn on/off monitoring, 93
features define with Cookie method, 187 test to assess if positive and significant, 213
file information displaying, 246
FTP results, 48
G
gene list type gal format file, 125 no gene list, 125, 129 tab text file, 125, 126
geometry information, 129 grid
hot key actions, 158 position main grid, 132 position spot centroids, 148 refit, 155 set up initial, 111 shortcuts for actions, 158 zoom in or out, 132
Grid Adjustment pane move main grid, 142 position spot centroids, 153 position subgrids, 144
grid editing change defaults, 157
grid file add to extraction set, 76 change in extraction set, 76 definition, 68 use if available, 50
grid mode access from extraction set, 115 access from image file, 117
grid mode, change default settings, 157
Feature Extraction for CytoGenomics 5.2 User Guide 275
grid placement allow some distortion, 175 failures, 98 place and rotate only, 175 summary report warnings, 98
grid template add to database, 76 add to extraction set, 75 change default protocol, 78 change in extraction set, 75 definition, 68 properties, 77
gridding if fails for Agilent image, 113
H
histograms changing bin size, 261 purpose, 263 selecting elliptical area, 263 selecting elliptical or rectangular region, 259 setting up, 259, 263 viewing, 263
hot key actions grid mode, 158 image analysis, 158
I
ignore spots, 152 image
cropping, 235 hot key actions, 267 printing, 265 saving cropped, 237, 238 viewing entire image, 257 viewing extraction results, 257
image display changing, 247
image orientation changing, 246
image, changing window size to fit, 257 initial grid, 111 intensity scale, changing, 252 IsPosAndSignif t-test, 213
276 Feature Extraction for CytoGenomics 5.2 User Guide
L
landscape, change to portrait, 123 line graphs, creating, 262 linear scale, changing to log, 252 local background
define with auto-estimate of local radius, 189 definition, 189
log ratios error models, 208 surrogates, 209
log scale, changing to linear, 252
M
magnification levels, 255 main grid
move position, 137 move with Grid Adjustment pane, 142 rotate position, 137
microarray information, process flow, 18 multiplicative error, 208
N
negative controls definition, 203
New Array Identifier, add or change, 239
O
orientation, portrait or landscape, 123 outliers
automatically compute polynomial terms, 195 features and local background, 193 interquartile range method, 194 non-uniformity, 194 parameters for GenePix protocol, 195 population, 194
output files GEML, 50 grid, 50 JPEG, 50 MAGE, 50 QC Report, 50
Feature Extraction for CytoGenomics 5.2 User Guide 277
text result, 50 visual results, 50
P
parameters for polynomial on, 195 pixel outliers
method to reject, 189 pixel saturation, 190 portrait, change to landscape, 123 position
main grid, 132 spot centroids, 148
Preferences dialog box changing other default settings, 265 grid mode, 157
print settings changing, 265
printing images, 265 process flow
microarray information, 18 project
change default properties, 53 change specific properties, 42 create new if run fails, 93, 114 default protocol, 50 folders, 55 load visual results, 101 on-time, 35 open, 40 save, 41 standard, 35 summary report, 97 terminate, 44
project properties differences between standard and on-time, 44
protocol 1-color, 79 1-color gene expression, 166 2-color Agilent gene expression, 162 2-color gene expression, 166 add new protocol, 170 add to extraction set, 87 Axon gene expression and CGH, 166 CGH, 162, 166 change in extraction set, 87 change in grid template, 78 change settings, 87
278 Feature Extraction for CytoGenomics 5.2 User Guide
change steps to run, 166, 170 create from existing protocol, 163 definition, 68 derived from, 165 export, 87 GenePix gene expression and CGH, 162 hidden settings, 163 import, 87 location for default, 49 new from template, 163 new, from template, 162 open, 164 project default, 50 properties, view or change, 165 removable from database?, 166 remove from database, 87 save as permanent read-only, 170 save to new name, 170 templates from Agilent, 162 type, 166 version, 165
protocol steps brief description, 167 calculate metrics, 227 compute ratios and errors, 222 correct dye biases, 216 find and measure spots, 182 flag outliers, 191 generate results, 230 place grid, 172
p-values surrogates, 209
Q
QC Report access from Project Run Summary, 98, 99 open, 99 print, 101 view, 99
QC Report metrics p-value, 229 replicate statistics, 228 spikein targets, 228
Feature Extraction for CytoGenomics 5.2 User Guide 279
R
radius method for background measurement, 189 rank consistency
use in dye normalization, 220 refit a grid, 155 replicate statistics
minimum population, 228 results
FTP to server, 60 jpeg compression factor, 231 loading visual, 240 overwrite, 51 save to folder, 48, 59 text output as single file, 230 text output as three files, 230 text result file, 95
Rosetta Resolver ACL settings, 47, 58 barcode for transfer, 99 data access settings, 47, 58 GEML transfer, 46, 57 MAGE transfer, 46, 57 send FTP results, 48
S
saturation definition, 190
saturation, pixels, 190 save grid file, 146 saving images
cropped, 237, 238 scan image properties, 246 shortcuts, find spots, 158 significance tests
2-sided t-test, 213 well above SD, 213
software features, 29 spike-in targets, 227 spot analysis
CookieCutter method, 187 WholeSpot method, 187
spot centroids calculate, 144 position, 148 position with Grid Adjustment pane, 153
280 Feature Extraction for CytoGenomics 5.2 User Guide
Spot Navigator, 150 spot size
calculate, 144 spots
calculate size, 182, 186 Cookie statistics method, 187 find and measure, 182 ignore, 152 statistics method to define features, 187 undo or redo position, 152 WholeSpot statistics method, 187
subgrid position with Grid Adjustment pane, 144 skew, 144
surrogates effect of use, 225
T
t-test IsPosAndSignif, 213 two-sided, 213
U
Universal Error Model additive error, 222 multiplicative error, 222
V
visual results load from a project, 101 view, 101
W
WholeSpot method, 187 window layout
default, 269 Grid Template Browser Pane, 269 Project Explorer Pane, 269 Project Work Window, 269 Protocol Browser Pane, 269
Feature Extraction for CytoGenomics 5.2 User Guide 281
restore, 271 set preferences, 271 when software starts, 271 Workspace, 269
windows and panes moving, 270
Z
zoom grid mode, 132
zooming tool and mouse buttons, 255
282 Feature Extraction for CytoGenomics 5.2 User Guide
www.agilent.com
In This Book
The User Guide presents instructions for using Agilent Feature Extraction for CytoGenomics software.
This guide provides instructions for:
Setting up and running Feature Extraction on batches of image files.
Creating grid files to assign to image files for automatic extraction.
Changing the parameter values for each step in a protocol that can be assigned to an image file for automatic extraction.
Working with image displays.
Related manuals for Agilent CytoGenomics 5.3 Software User Guide
en
Manualsnet FAQs
If you want to find out how the CytoGenomics 5.3 Agilent works, you can view and download the Agilent CytoGenomics 5.3 Software User Guide on the Manualsnet website.
Yes, we have the User Guide for Agilent CytoGenomics 5.3 as well as other Agilent manuals. All you need to do is to use our search bar and find the user manual that you are looking for.
The User Guide should include all the details that are needed to use a Agilent CytoGenomics 5.3. Full manuals and user guide PDFs can be downloaded from Manualsnet.com.
The best way to navigate the Agilent CytoGenomics 5.3 Software User Guide is by checking the Table of Contents at the top of the page where available. This allows you to navigate a manual by jumping to the section you are looking for.
This Agilent CytoGenomics 5.3 Software User Guide consists of sections like Table of Contents, to name a few. For easier navigation, use the Table of Contents in the upper left corner.
You can download Agilent CytoGenomics 5.3 Software User Guide free of charge simply by clicking the “download” button in the upper right corner of any manuals page. This feature allows you to download any manual in a couple of seconds and is generally in PDF format. You can also save a manual for later by adding it to your saved documents in the user profile.
To be able to print Agilent CytoGenomics 5.3 Software User Guide, simply download the document to your computer. Once downloaded, open the PDF file and print the Agilent CytoGenomics 5.3 Software User Guide as you would any other document. This can usually be achieved by clicking on “File” and then “Print” from the menu bar.